Compositions and methods for detecting gastrointestinal pathogens

Abstract

To provide compositions and methods for detecting gastrointestinal pathogens.SOLUTION: Compositions, methods, kits, and uses are provided for detecting the presence of Salmonella, Shigella, Campylobacter, and Shigatoxigenic Eschelichia coli (STEC) in samples. In some embodiments, the compositions, methods, kits, and uses comprise one or more oligonucleotides or the use of one or more oligonucleotides. In some embodiments, the method is carried out as a multiplex assay with separate primers / probes for C.jejuni and C.coli. Some of the oligonucleotides contain inosine and / or contain 5-methylcytosine substituted nucleotides. Described is the use of cyclodextrin and / or Polysorbate 20 in the amplification reactions.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 226,079, filed on Jul. 27, 2021, the disclosure of which is incorporated herein by reference in its entirety.

Background Art

[0002] Introduction and Summary Bacterial gastroenteritis is an inflammation of the gastrointestinal tract that causes acute diarrhea (episodes of three or more stools per day) lasting less than 14 days and may also include symptoms such as nausea, vomiting, and abdominal cramps. See Thielman and Guerrant, The New England Journal of Medicine, 350:38 - 47, 2004. In the United States, there are more than 200 million cases of diarrheal disease per year, resulting in an estimated 73 million physician visits, 1.8 million hospitalizations, and up to 6,000 deaths. See Guerrant et al., Clinical Infectious Diseases, 32:331 - 350, 2001. According to Centers for Disease Control Food Net data (compilation of data from 10 state health departments), in 2010, the number of reported infections and incidence per 100,000 population included: Salmonella (8,256, 17.6), Campylobacter (6,365, 13.6), and Shigella (1,780, 3.8). Centers for Disease Control and Prevention, “Vital Signs: Incidence and Trends of Infection with Pathogens Transmitted Commonly Through Food - Foodborne Diseases Active Surveillance Network, 10 U.S. Sites, 1996 - 2010,” MMWR See 60(22):749-755,2011. These three bacteria, along with Shigatoxigenic Escherichia coli (STEC), are common causes of bacterial gastroenteritis. The populations at highest risk for bacterial gastroenteritis infection are children (≤5), the elderly, and the immunocompromised. However, infections can occur in all age groups. The mode of infection is typically through ingestion of contaminated food or water, or via the fecal-oral route as a result of poor hygiene (handwashing).

[0003] Salmonella are Gram-negative, aerobic, rod-shaped bacilli. There are two species of Salmonella, including enterica and bongori. Salmonella enterica is further classified into six subspecies, and only a small part of Salmonella enterica subspecies I causes human disease. See Sabbagh et al., FEMS Microbiol Lett 305:1-13,2010. In the United States, Salmonella serotypes Typhimurium, Enteritidis, and Newport account for about half of the culture-confirmed Salmonella isolates. Salmonella serotype Typhi, which causes typhoid fever, is not common in the United States, but Salmonella serotypes Mississippi and Javiana are increasingly being identified as causes of disease. See Centers for Disease Control and Prevention, “Summary of Notifiable Diseases-United States,2008,” MMWR 57(54):15-16,2008.

[0004] Campylobacter are curved, motile, microaerophilic, Gram-negative rods. They exhibit rapid, darting motility in a corkscrew manner using one or two flagella and also possess lipopolysaccharide endotoxin. Two species of Campylobacter, C. jejuni and C. coli, are the cause of most human infections. See Klena et al., Journal of Clinical Microbiology, 42:5549-5557, 2004, Poly and Guerry, Current Opinion in Gastroenterology 24:27-31, 2008, Granato et al., Journal of Clinical Microbiology, 48:4022-4027, 2010.

[0005] E. coli is a Gram-negative rod commonly found in the lower intestine of warm-blooded organisms. Most strains of E. coli are non-pathogenic and are part of the normal intestinal flora. However, some strains, such as STEC, can cause life-threatening infections in humans. There are two main types of Shiga toxins produced by STEC, Shiga toxin type 1 and Shiga toxin type 2, carried by two different genes, stx1 and stx2, respectively. Some strains of STEC contain the stx1 gene, while other strains contain the stx2 gene. There are also specific STEC strains that contain both the stx1 and stx2 genes.

[0006] Shigella is a gram-negative, aerobic, rod-shaped bacterium that is closely related to E. coli. See Liu et al., FEMS Microbiol. Rev. 32:627-653, 2008. There are four species of Shigella, all of which cause disease in humans and can include S. sonnei (subgroup D), S. flexneri (subgroup B), S. boydii (subgroup B), and S. dysenteriae (subgroup A). According to the 2006 Shigella annual summary published by the CDC, S. sonnei is the most common cause of infection at 76%, followed by S. flexneri (14%), S. boydii (1.1%), and S. dysenteriae (0.5%). See Centers for Disease Control and Prevention, “Shigella Surveillance: Annual Summary, 2006,” Atlanta, GA: US Department of Health and Human Services, November 2008. There is a need to efficiently and sensitively detect the presence of Salmonella, Shigella, Campylobacter, and STEC in a sample, including a biological specimen, in order to provide diagnostic and prognostic information to physicians treating patients with or suspected of having bacterial gastroenteritis or related diseases.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0008] Accordingly, the present specification provides a set of oligonucleotides for determining the presence or absence of at least one enteric pathogen, comprising (a) to (e): (a) A Salmonella-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of (i) SEQ ID NO: 12 and SEQ ID NO: 14, (ii) SEQ ID NO: 21 and SEQ ID NO: 47, (iii) SEQ ID NO: 38 and SEQ ID NO: 36, (iv) SEQ ID NO: 35 and SEQ ID NO: 40, (v) SEQ ID NO: 12 and SEQ ID NO: 28, (vi) SEQ ID NO: 42 and SEQ ID NO: 31, or (vii) SEQ ID NO: 41 and SEQ ID NO: 27, (b) A C. jejuni-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 19, (c) A C. coli-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8 and SEQ ID NO: 10, (d) A Shigella-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of (i) SEQ ID NO: 15 and SEQ ID NO: 17, (ii) SEQ ID NO: 34 and SEQ ID NO: 22, or (iii) SEQ ID NO: 32 and SEQ ID NO: 33, and Provided is a set of oligonucleotides comprising at least one of Shigatoxigenic E. coli (STEC) - specific amplification oligomer sets, each of which comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of (e)(i) SEQ ID NO: 20 and SEQ ID NO: 3, (ii) SEQ ID NO: 49 and SEQ ID NO: 3, or (iii) SEQ ID NO: 4 and SEQ ID NO: 7.

[0009] In some embodiments, the set of oligonucleotides comprises a Salmonella - specific amplification oligomer set. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 47. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 38 and SEQ ID NO: 36. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 40. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 28. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 31. In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization array substantially corresponding to the nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 27.

[0010] In some embodiments, the set of oligonucleotides includes a C. jejuni - specific amplification oligomer set.

[0011] In some embodiments, the set of oligonucleotides includes a C. coli - specific amplification oligomer set.

[0012] In some embodiments, the set of oligonucleotides includes a Shigella - specific amplification oligomer set. In some embodiments, the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiments, the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 34 and SEQ ID NO: 22. In some embodiments, the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33.

[0013] In some embodiments, the set of oligonucleotides includes an STEC - specific amplification oligomer set.

[0014] In some embodiments, the set of oligonucleotides includes at least two of a Salmonella - specific amplification oligomer set, a C. jejuni - specific amplification oligomer set, a C. coli - specific amplification oligomer set, a Shigella - specific amplification oligomer set, and an STEC - specific amplification oligomer set.

[0015] In some embodiments, the set of oligonucleotides comprises at least three of a Salmonella - specific amplification oligomer set, a C. jejuni - specific amplification oligomer set, a C. coli - specific amplification oligomer set, a Shigella - specific amplification oligomer set, and an STEC - specific amplification oligomer set.

[0016] In some embodiments, the set of oligonucleotides comprises at least four of a Salmonella - specific amplification oligomer set, a C. jejuni - specific amplification oligomer set, a C. coli - specific amplification oligomer set, a Shigella - specific amplification oligomer set, and an STEC - specific amplification oligomer set.

[0017] In some embodiments, the set of oligonucleotides comprises a Salmonella - specific amplification oligomer set, a C. jejuni - specific amplification oligomer set, a C. coli - specific amplification oligomer set, a Shigella - specific amplification oligomer set, and an STEC - specific amplification oligomer set.

[0018] In some embodiments, the Salmonella - specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, the C. jejuni - specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 19, the C. coli - specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10, the Shigella - specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, The STEC-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of (i) SEQ ID NO: 20 and SEQ ID NO: 3, and (ii) SEQ ID NO: 4 and SEQ ID NO: 7.

[0019] In some embodiments, the set of oligonucleotides when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 13 when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 13 when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 45 when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 26, or SEQ ID NO: 25 when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 30 when the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 23, or When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 27 respectively, it further includes a Salmonella detection probe containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 43.

[0020] In some embodiments, in SEQ ID NO: 23, one or more, or each of nucleotides 2, 5, 7, and 14 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 25, one or more, or each of nucleotides 7, 9, 13, and 15 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 26, one or more, or each of nucleotides 9, 18, and 23 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 27, one or more, or each of nucleotides 6, 10, and 18 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 30, one or more, or each of nucleotides 2, 4, 18, 19, and 20 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 43, one or more, or each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 44, one or more, or each of nucleotides 7, 9, 13, and 15 is 5 - methylcytosine. In some embodiments, in SEQ ID NO: 45, one or more, or each of nucleotides 3, 6, 8, and 17 is 5 - methylcytosine.

[0021] In some embodiments, the set of oligonucleotides further includes a C. jejuni detection probe, and the C. jejuni detection probe contains a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 18.

[0022] In some embodiments, at SEQ ID NO: 18, one or more of nucleotides 7, 12, and 25, or each of nucleotides 7 and 12, or each of nucleotides 7, 12, and 25 is 5-methylcytosine.

[0023] In some embodiments, the set of oligonucleotides further comprises a C. coli detection probe, and the C. coli detection probe comprises a target hybridization sequence that substantially corresponds to the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 37, or SEQ ID NO: 39.

[0024] In some embodiments, at SEQ ID NO: 9, one or more or each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 is 5-methylcytosine. In some embodiments, at SEQ ID NO: 37, one or more or each of nucleotides 17, 22, 23, and 27 is 5-methylcytosine. In some embodiments, at SEQ ID NO: 39, one or more or each of nucleotides 2, 9, 12, 19, 22, 24, and 25 is 5-methylcytosine.

[0025] In some embodiments, the set of oligonucleotides further comprises a Shigella detection probe, and the Shigella detection probe SEQ ID NO: 16 when the Shigella-specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence that substantially corresponds to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 46 or SEQ ID NO: 29, or When the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33, it includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 24.

[0026] In some embodiments, at SEQ ID NO: 46, each of nucleotides 5, 11, and 12 is 5 - methylcytosine.

[0027] In some embodiments, the set of oligonucleotides further includes an STEC detection probe, and the STEC detection probe when the STEC - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 3, is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, when the STEC - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 49 and SEQ ID NO: 3, is SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, or when the STEC - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 7, includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0028] In some embodiments, in SEQ ID NO: 1, one or more, or each, of nucleotides 6, 7, 9, 14, 17, and 24 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 2, one or more, or each, of nucleotides 5, 8, 11, 13, 18, and 21 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 6, one or more, or each, of nucleotides 3, 7, 8, 15, 20, 24, and 27 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 15, one or more, or each, of nucleotides 7 and 15 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 19, one or more, or each, of nucleotides 1, 2, 7, 14, 15, 19, and 20 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 21, one or more, or each, of nucleotides 13, 17, and 24 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 28, one or more, or each, of nucleotides 2, 4, 12, 14, 15, and 17 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 31, one or more, or each, of nucleotides 6, 7, 8, 13, 23, 24, and 26 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 32, one or more, or each, of nucleotides 5, 11, 12, and 15 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 36, one or more, or each, of nucleotides 2, 3, 4, 16, 18, 19, and 22 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 38, one or more, or each, of nucleotides 4, 6, 7, and 8 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 40, one or more, or each, of nucleotides 4, 13, 18, and 22 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 47, one or more, or each, of nucleotides 11, 16, and 17 is 5-methylcytosine. In some embodiments, in SEQ ID NO: 7, one or more, or each, of nucleotides 3, 4, 18, and 19 is 5-methylcytosine.In some embodiments, at position SEQ ID NO: 12, one or more of nucleotides 4, 11, 12, and 16, or each is 5-methylcytosine. In some embodiments, at position SEQ ID NO: 22, each of nucleotides 5 and 16 is 5-methylcytosine.

[0029] In some embodiments, one or more, or each, of the detection probes comprises a fluorescent dye compound. In some embodiments, each of the detection probes further comprises a non-fluorescent quenching dye compound.

[0030] As used herein, an oligonucleotide for determining the presence or absence of at least one enteric pathogen, wherein the oligonucleotide is SEQ ID NO: 15 (wherein each of nucleotides 7 and 15 is 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (wherein each of nucleotides 1, 2, 7, 14, 15, 19, and 20 is 5-methylcytosine), SEQ ID NO: 21 (wherein each of nucleotides 13, 17, and 24 is 5-methylcytosine), SEQ ID NO: 23 (wherein each of nucleotides 2, 5, 7, and 14 is 5-methylcytosine), SEQ ID NO: 25 (wherein each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 26 (wherein each of nucleotides 9, 18, and 23 is 5-methylcytosine), SEQ ID NO: 27 (wherein each of nucleotides 6, 10, and 18 comprises 5-methylcytosine), SEQ ID NO: 28 (wherein each of nucleotides 2, 4, 12, 14, 15, and 17 is 5-methylcytosine), SEQ ID NO: 30 (wherein each of nucleotides 2, 4, 18, 19, and 20 is 5-methylcytosine), SEQ ID NO: 31 (wherein each of nucleotides 6, 7, 8, 13, 23, 24, and 26 is 5-methylcytosine), SEQ ID NO: 32 (wherein each of nucleotides 5, 11, 12, and 15 is 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 is 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 is 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 is 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 is 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 is 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 is 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 is 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 is 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 is 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 is 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 is 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 is 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 is 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 is 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 is 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 is 5-methylcytosine), and Also provided are oligonucleotides comprising any one of the sequences of SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 is 5-methylcytosine).

[0031] In some embodiments, the oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 is 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 is 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 is 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 is 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 is 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 is 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 is 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 is 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 is 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 is 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 is 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 is 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 is 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 is 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 is 5-methylcytosine), and SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 is 5-methylcytosine), and includes any one of the sequences.

[0032] This specification also provides a set of oligonucleotides of any one of the above embodiments or a kit containing any one of the oligonucleotides of the above embodiments.

[0033] This specification also provides a set of oligonucleotides of any one of the above embodiments or a reaction mixture containing any one of the oligonucleotides of the above embodiments.

[0034] This specification provides a multiplexing method, (1) contacting a sample suspected of containing at least one enteric pathogen with a set of oligonucleotides of any one of the previous embodiments; (2) performing an in vitro nucleic acid amplification reaction, which, if any Salmonella, C. jejuni, C. coli, Shigella, and STEC target nucleic acids are present in the sample, is used as a template to generate one or more amplification products corresponding to the Salmonella, C. jejuni, C. coli, Shigella, and STEC target regions; (3) detecting the presence or absence of one or more amplification products, Also provided is a multiplexing method including determining the presence or absence of at least one of Salmonella, C. jejuni, C. coli, Shigella, and STEC in the sample.

[0035] In some embodiments, step (3) of the multiplexing method is contacting the sample with at least one of a Salmonella detection probe, a C. jejuni detection probe, a C. coli detection probe, a Shigella detection probe, and an STEC detection probe, performing electrophoresis on the sample, or determining the sequence of one or more amplification products, if present.

[0036] Disclosed herein is a method for synthesizing an oligonucleotide, comprising (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position, (b) bonding the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue, (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues bonded to the solid support, (d) cleaving the at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining an oligonucleotide, wherein the oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 is 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 is 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 is 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 is 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 is 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 is 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 is 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 is 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 is 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 is 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 is 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 is 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 is 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 is 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 is 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 is 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 is 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 is 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 is 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 is 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 is 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 is 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 is 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 is 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 is 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 is 5-methylcytosine), and also provided is a method comprising any one of the sequences of SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 is 5-methylcytosine).

[0037] In some embodiments, the oligonucleotide has a length of 18 to 32 consecutive nucleic acid base residues. In some embodiments, the oligonucleotide has a length of 20 to 30 consecutive nucleic acid base residues.

[0038] Also provided herein is a method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, wherein each of synthesizing the first oligonucleotide and synthesizing the second oligonucleotide (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position; (b) bonding the 5'-position of the nucleobase residue furthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues attached to the solid support; (d) cleaving the at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining an oligonucleotide; and a first oligonucleotide and a second oligonucleotide each comprise SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 41 and SEQ ID NO: 27, SEQ ID NO: 11 and SEQ ID NO: 19, SEQ ID NO: 8 and SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 49 and SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 3, and a method comprising any one of the sequences of SEQ ID NO: 4 and SEQ ID NO: 7 is also provided.

[0039] In some embodiments, each of the first oligonucleotide and the second oligonucleotide has a length of 18 to 32 consecutive nucleobase residues. In some embodiments, each of the first oligonucleotide and the second oligonucleotide has a length of 20 to 30 consecutive nucleobase residues.

[0040] Therefore, the following embodiments are among those provided by the present disclosure.

[0041] Embodiment 1 is a set of oligonucleotides for determining the presence or absence of at least one enteric pathogen, comprising (a) to (e): (a) A Salmonella - specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of (i) SEQ ID NO: 12 and SEQ ID NO: 14, (ii) SEQ ID NO: 21 and SEQ ID NO: 47, (iii) SEQ ID NO: 38 and SEQ ID NO: 36, (iv) SEQ ID NO: 35 and SEQ ID NO: 40, (v) SEQ ID NO: 12 and SEQ ID NO: 28, (vi) SEQ ID NO: 42 and SEQ ID NO: 31, or (vii) SEQ ID NO: 41 and SEQ ID NO: 27; (b) A C. jejuni - specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 19; (c) A C. coli - specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8 and SEQ ID NO: 10; (d) A Shigella - specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of (i) SEQ ID NO: 15 and SEQ ID NO: 17, (ii) SEQ ID NO: 34 and SEQ ID NO: 22, or (iii) SEQ ID NO: 32 and SEQ ID NO: 33, and (e) A set of oligonucleotides comprising at least one of a Shigatoxigenic E. coli (STEC) - specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequence of (i) SEQ ID NO: 20 and SEQ ID NO: 3, (ii) SEQ ID NO: 49 and SEQ ID NO: 3, or (iii) SEQ ID NO: 4 and SEQ ID NO: 7.

[0042] Embodiment 2 is a set of oligonucleotides of Embodiment 1, which includes a Salmonella - specific amplification oligomer set.

[0043] Embodiment 3 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 47.

[0044] Embodiment 4 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 38 and SEQ ID NO: 36.

[0045] Embodiment 5 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 40.

[0046] Embodiment 6 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 28.

[0047] Embodiment 7 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 31.

[0048] Embodiment 8 is a set of oligonucleotides of Embodiment 2, wherein the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 27.

[0049] Embodiment 9 is a set of oligonucleotides of any one of the preceding embodiments, which includes a C. jejuni - specific amplification oligomer set.

[0050] Embodiment 10 is a set of oligonucleotides of any one of the preceding embodiments, which includes a C. coli - specific amplification oligomer set.

[0051] Embodiment 11 is a set of oligonucleotides of any one of the preceding embodiments, which includes a Shigella - specific amplification oligomer set.

[0052] Embodiment 12 is a set of oligonucleotides of Embodiment 11, wherein the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17.

[0053] Embodiment 13 is a set of oligonucleotides of Embodiment 11, wherein the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 34 and SEQ ID NO: 22.

[0054] Embodiment 14 is a set of oligonucleotides of Embodiment 11, wherein the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33.

[0055] Embodiment 15 is a set of oligonucleotides according to any one of the preceding embodiments, which includes an STEC-specific amplification oligomer set.

[0056] Embodiment 16 is a set of oligonucleotides according to any one of the preceding embodiments, which includes at least two of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set.

[0057] Embodiment 17 is a set of oligonucleotides according to any one of the preceding embodiments, which includes at least three of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set.

[0058] Embodiment 18 is a set of oligonucleotides according to any one of the preceding embodiments, which includes at least four of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set.

[0059] Embodiment 19 is a set of oligonucleotides according to any one of the preceding embodiments, which includes the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set.

[0060] Embodiment 20 is The Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14. The C. jejuni - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 19. The C. coli - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10. The Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17. The STEC - specific amplification oligomer set is a set of oligonucleotides of the immediately preceding embodiment, including a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of (i) SEQ ID NO: 20 and SEQ ID NO: 3 and (ii) SEQ ID NO: 4 and SEQ ID NO: 7.

[0061] Embodiment 21 is When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 13 When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 13 When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 45 When the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 26, or SEQ ID NO: 25, When the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 30, When the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 23, or When the Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 27, and further includes a Salmonella detection probe containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 43, it is a set of oligonucleotides of any one of Embodiments 2 to 20.

[0062] Embodiment 22 is a set of oligonucleotides of Embodiment 21, wherein in SEQ ID NO: 23, one or more of nucleotides 2, 5, 7, and 14, or each contains 5-methylcytosine.

[0063] Embodiment 23 is a set of oligonucleotides of Embodiment 21, wherein in SEQ ID NO: 25, one or more of nucleotides 7, 9, 13, and 15, or each contains 5-methylcytosine.

[0064] Embodiment 24 is a set of oligonucleotides of Embodiment 21, wherein in SEQ ID NO: 26, one or more of nucleotides 9, 18, and 23, or each contains 5-methylcytosine.

[0065] Embodiment 25 is a set of oligonucleotides of Embodiment 21, wherein at one or more of nucleotides 6, 10, and 18 in SEQ ID NO: 27, or each, contains 5-methylcytosine.

[0066] Embodiment 26 is a set of oligonucleotides of Embodiment 21, wherein at one or more of nucleotides 2, 4, 18, 19, and 20 in SEQ ID NO: 30, or each, contains 5-methylcytosine.

[0067] Embodiment 27 is a set of oligonucleotides of Embodiment 21, wherein at one or more of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 in SEQ ID NO: 43, or each, contains 5-methylcytosine.

[0068] Embodiment 28 is a set of oligonucleotides of Embodiment 21, wherein at one or more of nucleotides 7, 9, 13, and 15 in SEQ ID NO: 44, or each, contains 5-methylcytosine.

[0069] Embodiment 29 is a set of oligonucleotides of Embodiment 21, wherein at one or more of nucleotides 3, 6, 8, and 17 in SEQ ID NO: 45, or each, contains 5-methylcytosine.

[0070] Embodiment 30 is a set of oligonucleotides of any one of Embodiments 9 to 27, further comprising a C. jejuni detection probe, wherein the C. jejuni detection probe contains a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 18.

[0071] Embodiment 31 is a set of oligonucleotides of Embodiment 29, wherein at one or more of nucleotides 7, 12, and 25 in SEQ ID NO: 18, or each of nucleotides 7 and 12, or each of nucleotides 7, 12, and 25, contains 5-methylcytosine.

[0072] Embodiment 32 further includes a C. coli detection probe, and the C. coli detection probe includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 37, or SEQ ID NO: 39, and is a set of oligonucleotides of any one of Embodiments 10 to 30.

[0073] Embodiment 33 is a set of oligonucleotides of Embodiment 31, wherein in SEQ ID NO: 9, one or more of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29, or each contains 5-methylcytosine.

[0074] Embodiment 34 is a set of oligonucleotides of Embodiment 31, wherein in SEQ ID NO: 37, one or more of nucleotides 17, 22, 23, and 27, or each contains 5-methylcytosine.

[0075] Embodiment 35 is a set of oligonucleotides of Embodiment 31, wherein in SEQ ID NO: 39, one or more of nucleotides 2, 9, 12, 19, 22, 24, and 25, or each contains 5-methylcytosine.

[0076] Embodiment 36 further includes a Shigella detection probe, and the Shigella detection probe when the Shigella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 16, when the Shigella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 46 or SEQ ID NO: 29, or A set of oligonucleotides according to any one of Embodiments 11 to 35, wherein the Shigella-specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively, and further comprises a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 24.

[0077] Embodiment 37 is a set of oligonucleotides of Embodiment 36, wherein in SEQ ID NO: 46, each of nucleotides 5, 11, and 12 contains 5-methylcytosine.

[0078] Embodiment 38 further comprises an STEC detection probe, and the STEC detection probe when the STEC-specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 3, respectively, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, when the STEC-specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 49 and SEQ ID NO: 3, respectively, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, or when the STEC-specific amplification oligomer set comprises a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 7, respectively, it is a set of oligonucleotides according to any one of Embodiments 15 to 37, which contains a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0079] Embodiment 39 is a set of oligonucleotides of Embodiment 38, wherein in SEQ ID NO: 1, one or more, or each, of nucleotides 6, 7, 9, 14, 17, and 24 contains 5-methylcytosine.

[0080] Embodiment 40 is a set of oligonucleotides of Embodiment 38, wherein at one or more of nucleotides 5, 8, 11, 13, 18, and 21 in SEQ ID NO: 2, or each, contains 5-methylcytosine.

[0081] Embodiment 41 is a set of oligonucleotides of Embodiment 38, wherein at one or more of nucleotides 3, 7, 8, 15, 20, 24, and 27 in SEQ ID NO: 6, or each, contains 5-methylcytosine.

[0082] Embodiment 42 is a set of oligonucleotides of any one of the preceding embodiments, wherein at one or more of nucleotides 7 and 15 in SEQ ID NO: 15, or each, contains 5-methylcytosine.

[0083] Embodiment 43 is a set of oligonucleotides of any one of the preceding embodiments, wherein at one or more of nucleotides 1, 2, 7, 14, 15, 19, and 20 in SEQ ID NO: 19, or each, contains 5-methylcytosine.

[0084] Embodiment 44 is a set of oligonucleotides of any one of the preceding embodiments, wherein at one or more of nucleotides 13, 17, and 24 in SEQ ID NO: 21, or each, contains 5-methylcytosine.

[0085] Embodiment 45 is a set of oligonucleotides of any one of the preceding embodiments, wherein at one or more of nucleotides 2, 4, 12, 14, 15, and 17 in SEQ ID NO: 28, or each, contains 5-methylcytosine.

[0086] Embodiment 46 is a set of oligonucleotides of any one of the preceding embodiments, wherein at one or more of nucleotides 6, 7, 8, 13, 23, 24, and 26 in SEQ ID NO: 31, or each, contains 5-methylcytosine.

[0087] Embodiment 47 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 32, one or more of nucleotides 5, 11, 12, and 15, or each contains 5-methylcytosine.

[0088] Embodiment 48 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 36, one or more of nucleotides 2, 3, 4, 16, 18, 19, and 22, or each contains 5-methylcytosine.

[0089] Embodiment 49 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 38, one or more of nucleotides 4, 6, 7, and 8, or each contains 5-methylcytosine.

[0090] Embodiment 50 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 40, one or more of nucleotides 4, 13, 18, and 22, or each contains 5-methylcytosine.

[0091] Embodiment 51 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 47, one or more of nucleotides 11, 16, and 17, or each contains 5-methylcytosine.

[0092] Embodiment 52 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 7, one or more of nucleotides 3, 4, 18, and 19, or each contains 5-methylcytosine.

[0093] Embodiment 53 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 12, one or more of nucleotides 4, 11, 12, and 16, or each contains 5-methylcytosine.

[0094] Embodiment 54 is a set of oligonucleotides of any one of the preceding embodiments, wherein in SEQ ID NO: 22, each of nucleotides 5 and 16 contains 5-methylcytosine.

[0095] Embodiment 55 is a set of oligonucleotides of any one of Embodiments 21 to 54, wherein one or more of the detection probes, or each, contains a fluorescent dye compound.

[0096] Embodiment 56 is a set of oligonucleotides of the immediately preceding embodiment, wherein each of the detection probes further contains a non-fluorescent quenching dye compound.

[0097] Embodiment 57 is an oligonucleotide for determining the presence or absence of at least one enteric pathogen, wherein the oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 contains 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 contains 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 contains 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 contains 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 contains 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 contains 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 contains 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 contains 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 contains 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 contains 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 contains 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 contains 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 contains 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 contains 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 contains 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 contains 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 contains 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 contains 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 contains 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 contains 5-methylcytosine), and an oligonucleotide comprising any one of the sequences of SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 contains 5-methylcytosine).

[0098] Embodiment 58 is such that the oligonucleotide SEQ ID NO: 15 (each of nucleotides 7 and 15 contains 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 contains 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 contains 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 contains 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 contains 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 contains 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 contains 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 contains 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 contains 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 contains 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 contains 5-methylcytosine), and the oligonucleotide of the immediately preceding embodiment, comprising any one of the sequences of SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 contains 5-methylcytosine).

[0099] Embodiment 59 is a kit comprising a set of oligonucleotides of any one of Embodiments 1-56 or an oligonucleotide of Embodiment 57 or 58.

[0100] Embodiment 60 is a reaction mixture comprising a set of oligonucleotides of any one of Embodiments 1-55 or an oligonucleotide of Embodiment 57 or 58.

[0101] Embodiment 61 is the reaction mixture of the immediately preceding embodiment, further comprising α-cyclodextrin or polysorbate 20.

[0102] Embodiment 62 is the reaction mixture of Embodiment 60 or 61, further comprising α-cyclodextrin and polysorbate 20.

[0103] Embodiment 63 is the reaction mixture of Embodiment 61 or 62, wherein the concentration of α-cyclodextrin in the reaction mixture is from about 10 mg / mL to about 40 mg / mL.

[0104] Embodiment 64 is the reaction mixture of any one of Embodiments 61 - 63, wherein the concentration of α - cyclodextrin in the reaction mixture is about 16 mg / mL to about 30 mg / mL, about 15 mg / mL to about 20 mg / mL, or about 10 mg / mL to about 15 mg / mL.

[0105] Embodiment 65 is the reaction mixture of any one of Embodiments 61 - 64, wherein the concentration of α - cyclodextrin in the reaction mixture is about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL.

[0106] Embodiment 66 is the reaction mixture of any one of Embodiments 61 - 65, wherein the concentration of polysorbate 20 in the reaction mixture is about 0.002% to about 0.05% (v / v).

[0107] Embodiment 67 is the reaction mixture of any one of Embodiments 61 - 66, wherein the concentration of polysorbate 20 in the reaction mixture is about 0.003% to about 0.03% (v / v).

[0108] Embodiment 68 is the reaction mixture of any one of Embodiments 61 - 67, wherein the concentration of polysorbate 20 in the reaction mixture is about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v).

[0109] Embodiment 69 is the reaction mixture of any one of Embodiments 61 - 68, further comprising a detergent.

[0110] Embodiment 70 is the reaction mixture of the immediately preceding embodiment, wherein the detergent comprises sodium dodecyl sulfate.

[0111] Embodiment 71 is the reaction mixture of Embodiment 69 or 70, wherein the concentration of the detergent in the reaction mixture is about 3 mg / mL to 300 mg / mL.

[0112] Embodiment 72 is any one of the reaction mixtures of Embodiments 69 to 71, wherein the concentration of the detergent in the reaction mixture is from about 10 mg / mL to 100 mg / mL.

[0113] Embodiment 73 is any one of the reaction mixtures of Embodiments 69 to 72, wherein the concentration of the detergent in the reaction mixture is about 33.3 mg / mL.

[0114] Embodiment 74 is a multiplexing method comprising: (1) contacting a sample suspected of containing at least one enteric pathogen with any one set of oligonucleotides of Embodiments 1 to 56; (2) performing an in vitro nucleic acid amplification reaction, wherein if any Salmonella, C. jejuni, C. coli, Shigella, and STEC target nucleic acids are present in the sample, they are used as templates for generating one or more amplification products corresponding to the Salmonella, C. jejuni, C. coli, Shigella, and STEC target regions; (3) detecting the presence or absence of one or more amplification products, and thereby determining the presence or absence of at least one of Salmonella, C. jejuni, C. coli, Shigella, and STEC in the sample.

[0115] Embodiment 74.1 is the multiplexing method of the immediately preceding embodiment, wherein the in vitro nucleic acid amplification reaction occurs in any one of the reaction mixtures of Embodiments 60 to 73.

[0116] Embodiment 75 is the multiplexing method of Embodiment 74, wherein (3) comprises: contacting the sample with at least one of a Salmonella detection probe, a C. jejuni detection probe, a C. coli detection probe, a Shigella detection probe, and an STEC detection probe; performing electrophoresis on the sample; or A multiplexing method comprising determining the sequence of one or more amplification products, if present.

[0117] Embodiment 75.1 is the multiplexing method of the immediately preceding embodiment, wherein the in vitro nucleic acid amplification reaction occurs in the reaction mixture of any one of Embodiments 60 to 73.

[0118] Embodiment 76 is a method for synthesizing an oligonucleotide, comprising: (a) obtaining a solid support comprising at least one nucleobase residue, wherein at least one nucleobase residue is covalently bonded to the solid support at the 3'-position; (b) bonding the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues bound to the solid support; (d) cleaving at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining an oligonucleotide. The oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 contains 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 contains 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 contains 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 contains 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 contains 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 contains 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 contains 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 contains 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 contains 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 contains 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 contains 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 contains 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 contains 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 contains 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 contains 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 contains 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 contains 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 contains 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 contains 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 contains 5-methylcytosine), and SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 contains 5-methylcytosine),

[0119] Embodiment 77 is the method of Embodiment 76, wherein the oligonucleotide has a length of 18 to 32 consecutive nucleic acid base residues or 20 to 30 consecutive nucleic acid base residues.

[0120] Embodiment 78 is a method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, each of synthesizing the first oligonucleotide and synthesizing the second oligonucleotide comprising: (a) obtaining a solid support comprising at least one nucleic acid base residue, wherein the at least one nucleic acid base residue is covalently bonded to the solid support at the 3'-position; (b) bonding the 5'-position of the nucleic acid base residue furthest from the solid support to the 3'-position of another nucleic acid base residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleic acid base residues attached to the solid support; (d) cleaving the at least 15 consecutive nucleic acid base residues generated in step (c), thereby obtaining an oligonucleotide. The first oligonucleotide and the second oligonucleotide each correspond substantially to SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 41 and SEQ ID NO: 27, SEQ ID NO: 11 and SEQ ID NO: 19, SEQ ID NO: 8 and SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 49 and SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 3, and a method comprising any one of the sequences of SEQ ID NO: 4 and SEQ ID NO: 7.

[0121] Embodiment 79 is the method of Embodiment 78, wherein each of the first oligonucleotide and the second oligonucleotide has a length of 18 to 32 consecutive nucleic acid base residues, or 20 to 30 consecutive nucleic acid base residues. The present invention provides, for example, the following items. (Item 1) A set of oligonucleotides for determining the presence or absence of at least one enteric pathogen, comprising (a) to (e): (a) (i) A Salmonella-specific amplification oligomer set comprising first and second oligomers each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, (ii) SEQ ID NO: 21 and SEQ ID NO: 47, (iii) SEQ ID NO: 38 and SEQ ID NO: 36, (iv) SEQ ID NO: 35 and SEQ ID NO: 40, (v) SEQ ID NO: 12 and SEQ ID NO: 28, (vi) SEQ ID NO: 42 and SEQ ID NO: 31, or (vii) SEQ ID NO: 41 and SEQ ID NO: 27, (b)(i) A C. jejuni - specific amplification oligomer set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 19, (c)(i) A C. coli - specific amplification oligomer set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10, (d)(i) A Shigella - specific amplification oligomer set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, (ii) SEQ ID NO: 34 and SEQ ID NO: 22, or (iii) SEQ ID NO: 32 and SEQ ID NO: 33, and (e) A set of oligonucleotides comprising at least one of the Shigatoxigenic E. coli (STEC) - specific amplification oligomer sets, each set comprising a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of (i) SEQ ID NO: 20 and SEQ ID NO: 3, (ii) SEQ ID NO: 49 and SEQ ID NO: 3, or (iii) SEQ ID NO: 4 and SEQ ID NO: 7. (Item 2) The set of oligonucleotides according to Item 1, comprising the Salmonella - specific amplification oligomer set. (Item 3) The set of oligonucleotides according to any one of the preceding items, comprising the C. jejuni - specific amplification oligomer set. (Item 4) The set of oligonucleotides according to any one of the preceding items, comprising the C. coli - specific amplification oligomer set. (Item 5) The set of oligonucleotides according to any one of the preceding items, comprising the Shigella - specific amplification oligomer set. (Item 6) The set of oligonucleotides according to any one of the preceding items, comprising the STEC - specific amplification oligomer set. (Item 7) A set of oligonucleotides according to any one of the preceding items, comprising at least two of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set. (Item 8) A set of oligonucleotides according to any one of the preceding items, comprising at least three of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set. (Item 9) A set of oligonucleotides according to any one of the preceding items, comprising at least four of the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set. (Item 10) A set of oligonucleotides according to any one of the preceding items, comprising the Salmonella-specific amplification oligomer set, the C. jejuni-specific amplification oligomer set, the C. coli-specific amplification oligomer set, the Shigella-specific amplification oligomer set, and the STEC-specific amplification oligomer set. (Item 11) The Salmonella-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, The C. jejuni-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 19, The C. coli - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10, The Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, The STEC - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of (i) SEQ ID NO: 20 and SEQ ID NO: 3 and (ii) SEQ ID NO: 4 and SEQ ID NO: 7, the set of oligonucleotides described in the immediately preceding item. (Item 12) When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 13, When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 13, When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 45, When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 26, or SEQ ID NO: 25, When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 30, When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 23, or When the Salmonella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 27, and further includes a Salmonella detection probe containing a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 43, the set of oligonucleotides according to item 11. (Item 13) Further including a Shigella detection probe, wherein the Shigella detection probe When the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 16, When the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 46 or SEQ ID NO: 29, or When the Shigella - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33, and includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 24, the set of oligonucleotides according to item 11 or 12. (Item 14) Further including a STEC detection probe, wherein the STEC detection probe When the STEC - specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, When the STEC-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 49 and SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, or When the STEC-specific amplification oligomer set includes a first and a second oligomer each containing a target hybridization sequence substantially corresponding to the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 7, a set of oligonucleotides according to any one of items 11 to 13, which includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6. (Item 15) Further including a C. jejuni detection probe, wherein the C. jejuni detection probe includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 18, a set of oligonucleotides according to any one of items 3 to 14. (Item 16) Further including a C. coli detection probe, wherein the C. coli detection probe includes a target hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 37, or SEQ ID NO: 39, a set of oligonucleotides according to any one of items 4 to 15. (Item 17) A set of oligonucleotides according to any one of items 12 to 16, wherein one or more of the detection probes, or each, contains a fluorescent dye compound. (Item 18) A set of oligonucleotides according to the immediately preceding item, wherein each of the detection probes further contains a non-fluorescent quenching dye compound. (Item 19) An oligonucleotide for determining the presence or absence of at least one enteric pathogen, wherein the oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 contains 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 contains 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 contains 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 contains 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 contains 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 contains 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 contains 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 contains 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 contains 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 contains 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 contains 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 contains 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 contains 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 contains 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 contains 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 contains 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 contains 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 contains 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 contains 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 contains 5-methylcytosine), and an oligonucleotide comprising any one of the sequences of SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 contains 5-methylcytosine). (Item 20) A kit comprising the set of oligonucleotides according to any one of Items 1 to 18 or the oligonucleotide according to Item 19. (Item 21) A reaction mixture comprising the set of oligonucleotides according to any one of Items 1 to 18 or the oligonucleotide according to Item 19. (Item 22) The reaction mixture according to the immediately preceding item, further comprising α-cyclodextrin or polysorbate 20. (Item 23) The reaction mixture according to item 21 or 22, further comprising a detergent. (Item 24) The reaction mixture according to the immediately preceding item, wherein the detergent contains sodium dodecyl sulfate. (Item 25) A multiplexing method, comprising: (1) contacting a sample suspected of containing the at least one enteric pathogen with a set of oligonucleotides according to any one of items 1 to 56; (2) performing an in vitro nucleic acid amplification reaction, wherein, if any Salmonella, C. jejuni, C. coli, Shigella, and STEC target nucleic acids are present in the sample, they are used as templates for generating one or more amplification products corresponding to the Salmonella, C. jejuni, C. coli, Shigella, and STEC target regions; (3) detecting the presence or absence of the one or more amplification products, and thereby determining the presence or absence of at least one of Salmonella, C. jejuni, C. coli, Shigella, and STEC in the sample. (Item 26) (3) comprises contacting the sample with at least one of the Salmonella detection probe, the C. jejuni detection probe, the C. coli detection probe, the Shigella detection probe, and the STEC detection probe, performing electrophoresis on the sample, or determining the sequence of the one or more amplification products, if present, the multiplexing method according to the immediately preceding item. (Item 27) A method for synthesizing an oligonucleotide, comprising: (a) obtaining a solid support containing at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position; (b) binding the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues bound to the solid support; (d) cleaving the at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, wherein the oligonucleotide is SEQ ID NO: 15 (each of nucleotides 7 and 15 contains 5-methylcytosine), SEQ ID NO: 16, SEQ ID NO: 19 (each of nucleotides 1, 2, 7, 14, 15, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 21 (each of nucleotides 13, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 23 (each of nucleotides 2, 5, 7, and 14 contains 5-methylcytosine), SEQ ID NO: 25 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 26 (each of nucleotides 9, 18, and 23 contains 5-methylcytosine), SEQ ID NO: 27 (each of nucleotides 6, 10, and 18 contains 5-methylcytosine), SEQ ID NO: 28 (each of nucleotides 2, 4, 12, 14, 15, and 17 contains 5-methylcytosine), SEQ ID NO: 30 (each of nucleotides 2, 4, 18, 19, and 20 contains 5-methylcytosine), SEQ ID NO: 31 (each of nucleotides 6, 7, 8, 13, 23, 24, and 26 contains 5-methylcytosine), SEQ ID NO: 32 (each of nucleotides 5, 11, 12, and 15 contains 5-methylcytosine), SEQ ID NO: 36 (each of nucleotides 2, 3, 4, 16, 18, 19, and 22 contains 5-methylcytosine), SEQ ID NO: 38 (each of nucleotides 4, 6, 7, and 8 contains 5-methylcytosine), SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), SEQ ID NO: 40 (each of nucleotides 4, 13, 18, and 22 contains 5-methylcytosine), SEQ ID NO: 42, SEQ ID NO: 43 (each of nucleotides 2, 9, 12, 16, 17, 22, 23, and 26 contains 5-methylcytosine), SEQ ID NO: 44 (each of nucleotides 7, 9, 13, and 15 contains 5-methylcytosine), SEQ ID NO: 45 (each of nucleotides 3, 6, 8, and 17 contains 5-methylcytosine), SEQ ID NO: 47 (each of nucleotides 11, 16, and 17 contains 5-methylcytosine), SEQ ID NO: 1 (each of nucleotides 6, 7, 9, 14, 17, and 24 contains 5-methylcytosine), SEQ ID NO: 2 (each of nucleotides 5, 8, 11, 13, 18, and 21 contains 5-methylcytosine), SEQ ID NO: 6 (each of nucleotides 3, 7, 8, 15, 20, 24, and 27 contains 5-methylcytosine), SEQ ID NO: 7 (each of nucleotides 3, 4, 18, and 19 contains 5-methylcytosine), SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 contains 5-methylcytosine), SEQ ID NO: 12 (each of nucleotides 4, 11, 12, and 16 contains 5-methylcytosine), SEQ ID NO: 18 (each of nucleotides 7 and 12 or each of nucleotides 7, 12, and 25 contains 5-methylcytosine), SEQ ID NO: 22 (each of nucleotides 5 and 16 contains 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 contains 5-methylcytosine), and A method comprising any one of the sequences of SEQ ID NO: 46 (each of nucleotides 5, 11, and 12 contains 5-methylcytosine). (Item 28) The method according to item 27, wherein the oligonucleotide has a length of 18 to 32 consecutive nucleobase residues, or 20 to 30 consecutive nucleobase residues. (Item 29) A method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, wherein each of synthesizing the first oligonucleotide and synthesizing the second oligonucleotide (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position; (b) bonding the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues bonded to the solid support; (d) cleaving the at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide are each SEQ ID NO: 12 and SEQ ID NO: 14, SEQ ID NO: 21 and SEQ ID NO: 47, SEQ ID NO: 38 and SEQ ID NO: 36, SEQ ID NO: 35 and SEQ ID NO: 40, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 31, SEQ ID NO: 41 and SEQ ID NO: 27, SEQ ID NO: 11 and SEQ ID NO: 19, SEQ ID NO: 8 and SEQ ID NO: 10, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 49 and SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 3, and a method comprising any one of the sequences of SEQ ID NO: 4 and SEQ ID NO: 7. (Item 30) The method according to item 29, wherein each of the first oligonucleotide and the second oligonucleotide has a length of 18 to 32 consecutive nucleic acid base residues, or 20 to 30 consecutive nucleic acid base residues.

Mode for Carrying Out the Invention

[0122] Definitions Before explaining the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps and can therefore vary. As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an oligomer" includes plural oligomers and the like. The conjunction "or" should be interpreted in an inclusive sense, i.e., as equivalent to "and / or", unless an inclusive sense is unreasonable in the context.

[0123] It will be understood that, as used herein, the terms “about” or “substantially” are used in relation to properties such as temperature, concentration, time, etc., to indicate values within the scope of the teachings herein that are within a slight and very slight deviation. In general, the term “about” indicates a slight variation in the amount of a component of a composition that does not have any significant effect on the activity or stability of the composition. All ranges should be construed to include the endpoints thereof, unless an express exclusion such as “excluding the endpoints” is provided, and thus, for example, “within 10 to 15” includes the values 10 and 15. Also, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the teachings. To the extent that any incorporated materials by reference conflict with the explicit content of this disclosure, the explicit content prevails.

[0124] Unless otherwise specified, embodiments of the present specification that list various components as "comprising" are also assumed to "consist of" or "consist essentially of" the listed components. Embodiments of the present specification that list various components as "consisting of" are also assumed to "comprise" or "consist essentially of" the listed components. Embodiments of the present specification that list various components as "consisting essentially of" are also assumed to "consist of" or "comprise" the listed components (this interchangeability does not apply to the use of these terms in the claims). "Consisting essentially of" means that additional component(s), composition(s), or method step(s) that do not substantially change the basic and novel features of the compositions and methods described herein can be included in those compositions or methods. Such features include the ability to detect nucleic acid sequences of enteric pathogens present in a sample with a specificity that distinguishes the nucleic acid from other known pathogens, with a sensitivity that can optionally detect enteric pathogens at 75 - 150 CFU / mL, and optionally at about 90 - about 180 minutes or about 120 - about 150 minutes, and / or from the start of the amplification reaction when a cycle amplification reaction is used, at about 30 cycles - about 60 cycles, about 40 cycles - about 50 cycles, or about 45 cycles.

[0125] A "sample" or "specimen" that includes a "biological" or "clinical" sample refers to any material that may contain, or is suspected of containing, one or more of Salmonella, Shigella, Campylobacter jejuni, Campylobacter coli, and STEC, or a component thereof, such as a nucleic acid or a fragment of a nucleic acid. The sample can be a complex mixture of components. The sample includes a "biological sample" that includes any tissue or material derived from a living or dead mammal or organism, including, for example, feces, blood, plasma, serum, blood cells, saliva, mucosa, and cerebrospinal fluid. The sample can include feces from an organism that has experienced one or more symptoms of infection by one or more of Salmonella, Shigella, Campylobacter jejuni, Campylobacter coli, and STEC. The sample can also be a "spiked" sample, such as feces from an organism that has not experienced any symptoms of infection but in which one or more of Salmonella, Shigella, Campylobacter jejuni, Campylobacter coli, and STEC have been artificially added. The sample can also include a sample of in vitro cell culture components, such as a conditioned medium resulting from the growth of cells and tissues in a culture medium. To prepare a sample for analysis, the sample can be treated chemically, physically, or mechanically to disrupt the tissue or cell structure and release intracellular nucleic acids into a solution that may contain enzymes, buffers, salts, detergents, and the like. In one step of the methods described herein, a sample suspected of containing target nucleic acids of at least one enteric pathogen, such as Salmonella, Shigella, C. jejuni, C. coli, and STEC, is provided. Thus, this step excludes the physical step of obtaining the sample from a subject.

[0126] "Nucleic acid" and "polynucleotide" refer to multimeric compounds containing nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs joined together to form polynucleotides, including conventional RNA, DNA, hybrid RNA-DNA, and polymers that are analogs thereof. The nucleic acid "backbone" can be composed of various linkages including one or more of a sugar-phosphate diester linkage, a peptide-nucleic acid linkage ("peptide nucleic acid" or PNA, PCT No. 95 / 32305), a phosphorothioate linkage, a methylphosphonate linkage, or combinations thereof. The sugar moiety of the nucleic acid can be ribose, deoxyribose, or a similar compound having a substitution, for example, a 2'-methoxy or 2'-halide substitution. The nitrogenous bases are conventional bases (A, G, C, T, U), their analogs (e.g., inosine or others, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11 th ed., 1992), derivatives of purines or pyrimidines (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituents at the 5- or 6-position, purine bases having substituents at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4-alkyl-pyrimidine, U.S. Patent No. 5,378,825 and PCT No. 93 / 13121). The nucleic acid may contain one or more "abasic" residues where the backbone does not contain nitrogenous bases at the position(s) of the polymer (U.S. Patent No. 5,585,481). The nucleic acid may contain only conventional RNA or DNA sugars, bases, and linkages, or both conventional components and substitutions (e.g., a polymer containing both conventional bases with 2'-methoxy linkages or conventional bases and one or more base analogs). The nucleic acid contains "locked nucleic acid" (LNA), an analog containing one or more LNA nucleotide monomers that have bicyclic furanose units locked in an RNA-mimicking sugar structure and enhance hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Embodiments of oligomers that can affect the stability of the hybridization complex include oligomers containing PNA oligomers, 2'-methoxy or 2'-fluoro-substituted RNA, or oligomers containing charged linkages (e.g., phosphorothioate) or neutral groups (e.g., methylphosphonate), including oligomers that affect the total charge, charge density, or steric association of the hybridization complex. Unless otherwise indicated, 5-methylcytosine can be used with any of the aforementioned backbones / sugars / linkages including RNA or DNA backbones (or mixtures thereof). When referring to ranges for the length of an oligonucleotide, amplicon, or other nucleic acid, the range is understood to include all integers (e.g., a length of 19 to 25 consecutive nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0127] The "C" or "cytosine" residue includes methylated and non-methylated cytosine unless the context otherwise indicates.

[0128] "Oligonucleotide" or "oligomer" generally refers to a nucleic acid of less than 1,000 nucleotides (nt), including those having a size range with a lower limit of about 2 - 5 nt and an upper limit of about 500 - 900 nt. Some specific embodiments are oligonucleotides having a size range with a lower limit of about 5 - 15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50 - 600 nt, and other specific embodiments are in a size range having a lower limit of about 10 - 20 nt and an upper limit of about 22 - 100 nt. Oligonucleotides can be purified from naturally occurring sources but can be synthesized by using any well-known enzymatic or chemical method. Oligonucleotides can be referred to by a functional name (e.g., capture probe, primer, or promoter primer), and those skilled in the art will understand that such terms refer to oligomers.

[0129] "Amplicon" or "amplification product" means a nucleic acid molecule generated in a nucleic acid amplification reaction and derived from a target nucleic acid. The amplicon or amplification product contains a target nucleic acid sequence that can be the same as or in the opposite direction of the target nucleic acid.

[0130] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and is involved in a nucleic acid amplification reaction and functions, for example, as a primer or and a promoter - primer. Certain amplification oligonucleotides contain at least about 10 consecutive bases, optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive bases that are complementary to a region of the target nucleic acid sequence or its complementary strand. The consecutive bases can be at least about 80%, at least about 90%, or completely complementary to the target sequence to which the amplification oligomer binds. Those skilled in the art will understand that the recited ranges include all integers and rational numbers within the range (e.g., 92% or 98.377%). Certain amplification oligonucleotides are about 10 - about 60 bases in length and can optionally contain modified nucleotides.

[0131] "Primer" refers to an oligonucleotide having a 3' end that hybridizes to a template nucleic acid and is extended by polymerization. The primer can be optionally modified, for example, by including a 5' region that is non-complementary to the target sequence. Such modifications can include the addition of functionality, such as tags, promoters, or other sequences that are used to manipulate or amplify the primer or target oligonucleotide or are otherwise useful.

[0132] In the context of transcription-mediated amplification, a primer modified with a 5' promoter sequence can be referred to as a "promoter-primer". One of ordinary skill in the art of molecular biology or biochemistry will understand that an oligonucleotide that can function as a primer can be modified to include a 5' promoter sequence and function as a promoter-primer, and similarly, any promoter-primer can function as a primer that includes or does not include its 5' promoter sequence.

[0133] "Nucleic acid amplification" refers to any in vitro procedure that produces multiple copies of a target nucleic acid sequence, or its complementary sequence, or fragments thereof (i.e., an amplified sequence containing less than the full target nucleic acid). Examples of nucleic acid amplification procedures include transcription-related methods, such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and others (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), ligase chain reaction (LCR) (e.g., European Patent Application No. 0320308), helicase-dependent amplification (e.g., U.S. Pat. No. 7,282,328), and strand displacement amplification (SDA) (e.g., U.S. Pat. No. 5,422,252). Amplification can be linear or exponential. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB-replicase. PCR amplification uses a DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of the two complementary strands of DNA or cDNA. LCR amplification uses at least four separate oligonucleotides and multiple cycles of hybridization, ligation, and denaturation to amplify the target and its complementary strand. Helicase-dependent amplification uses a helicase to separate the two strands of a DNA duplex to generate a single-stranded template, followed by hybridization of sequence-specific primers that hybridize to the template and extension by a DNA polymerase that amplifies the target sequence. SDA uses a primer that contains a recognition site for a restriction endonuclease that will nick one strand of a hemimodified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Particular embodiments use PCR or TMA, but it will be apparent to those skilled in the art that the oligonucleotides disclosed herein can be readily used as primers in other amplification methods.

[0134] Transcription-mediated amplification uses DNA polymerase, RNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-containing oligonucleotide and may optionally include other oligonucleotides to ultimately produce multiple RNA transcripts from a nucleic acid template (described in U.S. Patent Nos. 5,399,491 and 5,554,516, Kacian et al., U.S. Patent No. 5,437,990, Burg et al., PCT Nos. 88 / 01302 and 88 / 10315, Gingeras et al., U.S. Patent No. 5,130,238, Malek et al., U.S. Patent Nos. 4,868,105 and 5,124,246, Urdea et al., PCT No. 94 / 03472, McDonough et al., PCT No. 95 / 03430, and Ryder et al.). Methods using TMA have been previously described in detail (U.S. Patent Nos. 5,399,491 and 5,554,516).

[0135] In cyclic amplification methods that detect amplicons in real time, the term "threshold cycle" (Ct) is a measure of the time of appearance of a signal associated with amplification of a target and is generally 10 times the standard deviation of the normalized reporter signal. When amplification reaches the "threshold cycle", it is generally considered that there is a positive amplification product of the sequence to which the probe binds. The identity of the amplification product can then be determined by methods known to those of skill in the art, such as gel electrophoresis, nucleic acid sequencing, and other such analytical procedures.

[0136] As used herein, the term "relative fluorescence unit" ("RFU") is a unit of measurement of fluorescence intensity. RFU varies with the characteristics of the detection means used for the measurement and can be used as a measured value to compare the relative intensities between a sample and a control.

[0137] A "detection probe" or "probe" refers to an oligonucleotide that specifically hybridizes to a target sequence, including an amplified sequence, under conditions that promote nucleic acid hybridization for the detection of a target nucleic acid. Detection can be either direct (i.e., a probe that hybridizes directly to the target) or indirect (i.e., a probe that hybridizes to an intermediate structure that binds the probe to the target). The target sequence of the probe generally refers to a specific sequence within a larger sequence to which the probe specifically hybridizes. A detection probe can include a target-specific sequence and a non-target complementary sequence. Such non-target complementary sequences can include sequences that confer a desired secondary or tertiary structure, such as a hairpin structure, that can be used to facilitate detection and / or amplification (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412). Probes of defined sequences can be produced by techniques known to those of skill in the art, for example, by chemical synthesis and by in vitro or in vivo expression from recombinant nucleic acid molecules.

[0138] As used herein, a nucleic acid that "substantially corresponds" to a designated nucleic acid sequence, or its complement, means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide will hybridize to the same target nucleic acid sequence under stringent hybridization conditions, in terms of having hybridization characteristics similar to the reference nucleic acid sequence. A substantially corresponding nucleic acid differs from a particular nucleic acid by at least one nucleotide. This variation can be described in terms of the percentage of sequence identity or complementarity between the nucleic acid and the particular nucleic acid (e.g., less than 100% to about 80%). One of skill in the art will understand that the recited ranges include all integers and rational numbers within the range (e.g., 92%, 92.377%, etc.).

[0139] "Hybridization" or "hybridize" means the ability of two completely or partially complementary nucleic acid strands, in parallel or antiparallel orientation, to come together under specific hybridization assay conditions and form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. These hydrogen bonds are most commonly formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid strand, although base pairing can also occur between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, for example, Adams et al., The Biochemistry of the Nucleic Acids (11th ed. 1992).)

[0140] "Preferentially hybridize" means that under stringent hybridization conditions, an amplification or detection probe oligonucleotide can hybridize to its target nucleic acid and form a stable oligonucleotide:target hybrid, but does not form a sufficient number of stable oligonucleotide:non-target hybrids. Amplification and detection oligonucleotides that preferentially hybridize to a target nucleic acid are useful for amplifying and detecting the target nucleic acid, but not for non-target organisms, especially those that are phylogenetically closely related. Thus, the oligonucleotide hybridizes to the target nucleic acid to a sufficiently greater extent than to non-target nucleic acids to enable one of ordinary skill in the art to accurately amplify and / or detect the presence (or absence) of nucleic acids derived from a specified enteric pathogen, as needed. In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will decrease the degree or proportion of hybridization of the oligonucleotide to its target region. However, the inclusion of one or more non-complementary nucleosides or nucleic acid bases can enhance the ability of the oligonucleotide to discriminate against non-target organisms.

[0141] Preferred hybridization is known in the art and can be measured using techniques described herein, for example, in the examples provided below. In some embodiments, there is at least a 10-fold difference, at least a 100-fold difference, or at least a 1,000-fold difference between the target and non-target hybridization signals in the test sample. In some embodiments, the non-target hybridization signal in the test sample is below the background signal level.

[0142] "Stringent hybridization conditions" or "stringent conditions" mean conditions that allow an oligomer to preferentially hybridize to a target nucleic acid (such as an enteric pathogen nucleic acid), but do not allow preferential hybridization to nucleic acids derived from closely related non-target nucleic acids. The definition of stringent hybridization conditions does not vary, but the actual reaction environment that can be used for stringent hybridization can vary depending on the GC content and length of the oligonucleotide, the degree of similarity between the oligonucleotide sequence and the sequences of non-target nucleic acids that may be present in the test sample, and factors including the target sequence. Hybridization conditions include the temperature and composition of the hybridization reagent or solution. Exemplary hybridization assay conditions for amplifying and / or detecting target nucleic acids derived from one or more target enteric pathogens with the oligonucleotides of the present disclosure correspond to a temperature of about 60°C when the salt concentration of a monovalent salt, such as KCl, is in the range of about 0.06 - 0.09 M. Specific hybridization assay conditions are described in the Examples section below. Other acceptable stringent hybridization conditions can be readily identified by those skilled in the art.

[0143] "Assay conditions" mean conditions that allow stable hybridization of an oligonucleotide to a target nucleic acid. Assay conditions do not require preferential hybridization of the oligonucleotide to the target nucleic acid.

[0144] "Label" or "detectable label" refers to a moiety or compound that is directly or indirectly conjugated to a probe that yields a detectable or detectable signal. Direct conjugation may use covalent bonds or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and chelate or coordination complex formation), while indirect conjugation may use a bridging moiety or linker that amplifies the detectable signal (e.g., via an antibody or additional oligonucleotide(s)). Any detectable moiety can be used, such as a radionuclide, a ligand such as biotin or avidin, an enzyme, an enzyme substrate, a reactive group, a chromophore such as a dye or particle that confers a detectable color (e.g., latex or metal beads), a luminescent compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent compound), and a fluorescent compound (i.e., a fluorophore). Embodiments of fluorophores include those that absorb light in the range of about 495 - 690 nm and emit light in the range of about 520 - 705 nm, including those known as FAM™, TET™, CAL FLUOR™ (Orange or Red), and QUASAR™ compounds. Fluorophores can be used in combination with quencher molecules that absorb light and reduce background fluorescence when in proximity to the fluorophore. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER™ (or BHQ™) or TAMRA™ compounds. Certain embodiments include "homogeneous detectable labels" that are detectable in a homogeneous system in which the bound labeled probe in a mixture exhibits a detectable change compared to the unbound labeled probe, which allows the label to be detected without physically removing the hybridized labeled probe from the unhybridized labeled probe (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Certain homogeneous detectable labels include chemiluminescent compounds, including acridinium ester ("AE") compounds, such as the well-known, standard AE or AE derivatives (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604).Methods for synthesizing labels, methods for binding labels to nucleic acids, and methods for detecting signals from labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd. ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) Chapter 10, as well as U.S. Patent Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, and European Patent Application No. 0747706). Specific methods for binding AE compounds to nucleic acids are known (see, e.g., U.S. Patent Nos. 5,585,481 and 5,639,604, column 10, line 6 to column 11, line 3, and Example 8). Specific AE label positions are in the central region of the probe and in the vicinity of the region of A / T base pairs, at the 3' or 5' end of the probe, or at or near a mismatch site with a known sequence that the probe should not be detected compared to the desired target sequence. Other detectably labeled probes include TaqMan™ probes, molecular torches, and molecular beacons. TaqMan™ probes contain donor and acceptor labels, and fluorescence is detected when the probe is enzymatically degraded during amplification to release the fluorophore from the quencher. Molecular torches and beacons exist in open and closed configurations, the closed configuration quenches the fluorophore, and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to the target opens the otherwise closed probe.

[0145] "Non-extendable" oligonucleotides contain a blocking moiety at or near their 3'-end to prevent extension. The blocking group near the 3'-end is, in some embodiments, within 5 residues of the 3'-end and large enough to limit binding of the polymerase to the oligomer. Other embodiments contain a blocking group covalently attached to the 3'-end. Many different chemical groups, such as alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, and cordycepin, can be used to block the 3'-end. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that interfere with extension; inverted nucleotides (e.g., bound to the preceding nucleotide through a 3'-~-3' phosphodiester with an optionally exposed 5'-OH or phosphate); or proteins or peptides bound to the oligonucleotide to prevent further extension of the nascent nucleic acid strand by the polymerase. The non-extendable oligonucleotides of the present disclosure can be at least 10 bases in length and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Non-extendable oligonucleotides containing a detectable label can be used as probes.

[0146] In particular, references to "the sequence of SEQ ID NO: X" in the claims refer to the nucleotide sequence of the corresponding Sequence Listing entry, unless otherwise specifically indicated, and do not require identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues).

[0147] "Non-Watson-Crick" (NWC) positions in an oligonucleotide refer to positions that are configured such that the oligonucleotide hybridizes to at least one target sequence in a non-Watson-Crick pairing, including those due to the use of inosine (e.g., via hypoxanthine (I) of inosine). Such NWC pairings include, for example, I-A, I-T, I-C, I-G, I-U, G-U, G-T, and G-A (any of I / A / T / C / G / U can be a base in the oligonucleotide). In some embodiments, the NWC position is configured to hybridize via an I-A pair. In some embodiments, the NWC position is configured to hybridize via an I-T pair. In some embodiments, the NWC position is configured to hybridize via an I-C pair. In some embodiments, the NWC position is configured to hybridize via an I-G pair. In some embodiments, the NWC position is configured to hybridize via an I-U pair. In some embodiments, the NWC position is configured to hybridize via a wobble (G-U) or purine-purine (G-A) pair. In some embodiments, the NWC position is configured to hybridize via a G-T pair. In some embodiments, the NWC position is configured to hybridize via a G-U pair. In some embodiments, the NWC position is configured to hybridize via a G-A pair.

[0148] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. General definitions can be found in technical books related to the field of molecular biology, such as DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, NY) or THE HARPER COLLINS DICTIONARY OF BIOLOGY (Hale & Marham, 1991, Harper Perennial, New York, NY).

[0149] Exemplary Compositions, Kits, Methods, and Uses The present disclosure provides oligomers, compositions, and kits useful for determining the presence or absence of at least one enteric pathogen, such as Salmonella, C. jejuni, C. coli, Shigella, and STEC, in a sample.

[0150] In some embodiments, the oligonucleotides are provided, for example, in a kit or composition. The oligonucleotides generally include a target hybridization region configured to specifically hybridize to a target nucleic acid of an enteric pathogen. Oligonucleotides of different lengths and base compositions can be used to amplify the target nucleic acid, but in some embodiments, the oligonucleotides of the present disclosure have a target hybridization region that is about 10 to 60 bases in length, about 14 to 50 bases in length, about 14 to 40 bases in length, about 14 to 35 bases in length, or about 15 to 30 bases in length. In some embodiments, the oligonucleotide includes a region of a second sequence, such as a promoter, that can be located 5' to the target hybridization region, in addition to the target hybridization region. In some embodiments, the oligonucleotide does not include a region of a second sequence.

[0151] In some embodiments, a set of oligonucleotides is provided, wherein one oligonucleotide is configured to hybridize to the sense strand of the target nucleic acid and the other oligonucleotide is configured to hybridize to the antisense strand of the target nucleic acid. Such oligonucleotides include an amplification oligomer set (e.g., a primer pair) for PCR or other forms of amplification.

[0152] In some embodiments, one or more oligonucleotides, such as primer pairs or primer pairs and optionally a third oligonucleotide (for use as a probe, for example) that is optionally labeled, are configured to hybridize to a target nucleic acid of one or more enteric pathogens, including Salmonella, C. jejuni, C. coli, Shigella, and STEC. In some embodiments, multiple oligonucleotides, such as multiple primer pairs or multiple primer pairs and optionally a third oligonucleotide (for use as a probe, for example) that is optionally labeled, are configured to collectively hybridize to one or more target nucleic acids of one or more enteric pathogens, including Salmonella, C. jejuni, C. coli, Shigella, or STEC.

[0153] In some embodiments, one or more oligonucleotides include non-Watson-Crick (NWC) positions. In some embodiments, Salmonella primers, Salmonella primer pairs, and / or Salmonella probes include NWC positions, such as positions that include inosine. In some embodiments, C. jejuni primers, C. jejuni primer pairs, and / or C. jejuni probes include NWC positions, such as positions that include inosine. In some embodiments, C. coli primers, C. coli primer pairs, and / or C. coli probes include NWC positions, such as positions that include inosine. In some embodiments, Shigella primers, Shigella primer pairs, and / or Shigella probes include NWC positions, such as positions that include inosine. In some embodiments, STEC primers, STEC primer pairs, and / or STEC probes include NWC positions, such as positions that include inosine.

[0154] In some embodiments, one or more oligonucleotides include positions that include 5-methylcytosine. In some embodiments, Salmonella primers, Salmonella primer pairs, and / or Salmonella probes include positions that include 5-methylcytosine. In some embodiments, C. jejuni primers, C. jejuni primer pairs, and / or C. jejuni probes include positions that include 5-methylcytosine. In some embodiments, C. coli primers, C. coli primer pairs, and / or C. coli probes include positions that include 5-methylcytosine. In some embodiments, Shigella primers, Shigella primer pairs, and / or Shigella probes include positions that include 5-methylcytosine. In some embodiments, STEC primers, STEC primer pairs, and / or STEC probes include positions that include 5-methylcytosine.

[0155] In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 12 and SEQ ID NO: 14. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 21 and SEQ ID NO: 47. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 38 and SEQ ID NO: 36. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 35 and SEQ ID NO: 40. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 12 and SEQ ID NO: 28. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 42 and SEQ ID NO: 31. In some embodiments, the first and second oligomers of the Salmonella-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 41 and SEQ ID NO: 27.

[0156] In some embodiments, the first and second oligomers of the C. jejuni-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 11 and SEQ ID NO: 19.

[0157] In some embodiments, the first and second oligomers of the C. jejuni-specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 11 and SEQ ID NO: 19.

[0158] In some embodiments, the first and second oligomers of the C. coli - specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 8 and SEQ ID NO: 10.

[0159] In some embodiments, the first and second oligomers of the C. coli - specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 8 and SEQ ID NO: 10.

[0160] In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 34 and SEQ ID NO: 22. In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 32 and SEQ ID NO: 33.

[0161] In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 34 and SEQ ID NO: 22. In some embodiments, the first and second oligomers of the Shigella - specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 32 and SEQ ID NO: 33.

[0162] In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 20 and SEQ ID NO: 3. In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 49 and SEQ ID NO: 3. In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each comprise a target hybridization sequence comprising SEQ ID NO: 4 and SEQ ID NO: 7.

[0163] In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 20 and SEQ ID NO: 3. In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 49 and SEQ ID NO: 3. In some embodiments, the first and second oligomers of the STEC-specific amplification oligomer set each consist of a target hybridization sequence comprising SEQ ID NO: 4 and SEQ ID NO: 7.

[0164] Exemplary primer pairs and optional third oligomers (e.g., probes) are set forth in Table A below.

[0165] [Table A]

[0166] In some embodiments, oligonucleotides comprising a label are provided. Such oligonucleotides can be used as probes. In some embodiments, the labeled oligonucleotides have sequences corresponding to the SEQ ID NOS. listed in the 3rd column of Oligomers in Table A. In some embodiments, the label is a non-nucleotide label. Suitable labels include compounds that emit a detectable optical signal, such as fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. Two or more labels, and two or more types of labels, can be present on a particular probe, or detection can rely on using a mixture of probes, each of which is labeled with a compound that produces a detectable signal (see, e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). The label can be attached to the probe by various means including covalent bonding, chelation, and ionic interactions, but in some embodiments, the label is covalently attached. For example, in some embodiments, the detection probe has a conjugated chemiluminescent label, such as an acridinium ester (AE) compound (see, e.g., U.S. Pat. Nos. 5,185,439, 5,639,604, 5,585,481, and 5,656,744). Labels, such as fluorescent or chemiluminescent labels, can be attached to the probe by a non-nucleotide linker (c Pat. Nos. 5,585,481, 5,656,744, and 5,639,604). In some embodiments, the label can include one or more of Quasar670, CalRed610, CalOrange560, fluorescein, ROX, FAM, and HEX.

[0167] In some embodiments, a probe (e.g., including a fluorescent label) further includes a second label that interacts with the first label. For example, the second label can be a quencher. In some embodiments, the second label can include one or both of BHQ-1 and BHQ-2. Such probes can be used, for example, in TaqMan™ assays, where hybridization of the probe to a target or amplicon of the probe, followed by nuclease digestion by a polymerase with 5'-3' exonuclease activity, results in release of the fluorescent label and thereby increased fluorescence, or fluorescence independent of the interaction with the second label.

[0168] In some applications, one or more probes that exhibit at least some degree of self-complementarity are used to facilitate the detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probes. Specific embodiments of such detection probes include probes that form a three-dimensional structure retained by intramolecular hybridization, such as a three-dimensional structure commonly referred to as a hairpin. Suitable hairpin probes include "molecular torches" (see, e.g., U.S. Patent Nos. 6,849,412, 6,835,542, 6,534,274, and 6,361,945) and "molecular beacons" (see, e.g., U.S. Patent Nos. 5,118,801 and 5,312,728). A molecular torch includes separate regions of self-complementarity (engineered "target binding domains" and "target closing domains") that are connected by a binding region (e.g., a -(CH2CH2O)3 - linker) and hybridize to each other under predetermined hybridization assay conditions. When exposed to appropriate target or denaturing conditions, the two complementary regions of the molecular torch (which may be fully or partially complementary) melt, making the target binding domain available for hybridization to a target sequence when the predetermined hybridization assay conditions are restored. The molecular torch is designed such that the target binding domain has a preference for hybridization to the target sequence over the target closing domain. The target binding domain and target closing domain of the molecular torch include interaction labels (e.g., fluorescence / quencher) positioned such that different signals are produced when the molecular torch is self-hybridized as opposed to when the molecular torch is hybridized to a target nucleic acid, thereby enabling the detection of probe:target duplexes in a test sample in the presence of unhybridized probes having an associated detectable label.

[0169] Examples of interacting donor / acceptor label pairs that can be used in connection with the present disclosure, without attempting to distinguish FRET from non-FRET pairs, include fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, lucifer yellow / DABCYL, BODIPY / DABCYL, eosin / DABCYL, erythrosin / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BHQ-1, CY5 / BHQ-2, CY3 / BHQ-1, CY3 / BHQ-2, and fluorescein / QSY7 dyes. One of ordinary skill in the art will understand that when the donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of the donor fluorescence. Non-fluorescent acceptors such as DABCYL and QSY7 dyes advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e., non-sensitized) acceptor excitation. Exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and CY dyes (such as CY5). Exemplary quencher moieties that can be used as the other member of a donor-acceptor pair include DABCYL and Black Hole Quencher moieties available from Biosearch Technologies, Inc. (Novato, Calif.).

[0170] In some embodiments, the labeled oligonucleotide (e.g., probe) is non-extendable. For example, the labeled oligomer can be made non-extendable by having 3'-phosphorylation, 3'-terminal 3'-deoxynucleotides (e.g., terminal 2',3'-dideoxynucleotides), having 3'-terminal inverted nucleotides (e.g., the last nucleotide is inverted such that it is linked to the second last nucleotide by a 3'-3' phosphodiester bond or an analog thereof, such as phosphorothioate), or having a linked fluorophore, quencher, or other label that interferes with extension (possibly linked via the 3'-position of the terminal nucleotide, but not necessarily). In some embodiments, the 3'-terminal nucleotide is not methylated.

[0171] The present disclosure also provides a reaction mixture for determining the presence or absence of, or quantifying the amount of, a target nucleic acid of at least one enteric pathogen, such as Salmonella, C. jejuni, C. coli, Shigella, and STEC, in a sample. A reaction mixture according to the present disclosure comprises at least one or more of the oligonucleotides described herein for amplification of a target nucleic acid and the oligonucleotides (e.g., probes) described herein for determining the presence or absence of an amplification product of the target nucleic acid. For a reaction mixture comprising a detection probe together with an amplification oligonucleotide combination, the amplification oligonucleotide and the detection probe oligonucleotide for the reaction mixture are bound by a common target region (i.e., the reaction mixture will comprise a probe that binds to a sequence amplifiable by the amplification oligonucleotide combination of the reaction mixture).

[0172] The reaction mixture may further comprise a number of optional components, such as capture probes, e.g., poly-(k) capture probes as described in US2013 / 0209992, incorporated herein by reference, and / or poly-(R) capture probes as described in US2020 / 0165599, incorporated herein by reference. For an amplification reaction mixture, the reaction mixture typically contains other reagents suitable for performing in vitro amplification, such as, for example, a buffer, a salt solution, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP, and / or ATP, CTP, GTP, and UTP), and / or an enzyme (e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or an RNA polymerase), and will typically contain a test sample component, whether or not the target nucleic acid is present. Suitable reagents include, for example, formulations containing lithium lauryl sulfate (LLS), sodium lauryl sulfate (SLS), NaH2PO4, Na2HPO4, EDTA, EGTA, LiOH, NaCl, KCl, MgCl2, NaOH, ethanol, methylparaben, propylparaben, trehalose, Tris Buffer, Triton® X-100, paramagnetic particles, target capture oligonucleotides, HEPES, succinic acid, polymerases (e.g., DNA polymerase, reverse transcriptase), and / or RNasin.

[0173] In some embodiments, the reaction mixture contains KCl. In some embodiments, the KCl concentration is about 50 mM. In some embodiments, the KCl concentration is greater than about 50 mM, for example, about 60 - 150 mM, about 75 - 125 mM, about 80 - 120 mM, about 85 - 115 mM, or about 90 - 110 mM. In some embodiments, the KCl concentration is 55 - 65, 65 - 75, 75 - 85, 85 - 95, 95 - 105, 105 - 115, 115 - 125, 125 - 135, or 135 - 145, each of which is in mM and optionally modified by "about". In some embodiments, the compositions according to the present disclosure contain KCl at any of the aforementioned concentrations, for example. In some embodiments, the methods according to the present disclosure include performing an amplification reaction in the presence of KCl at any of the aforementioned concentrations, for example.

[0174] In some embodiments, the reaction mixture comprises α-cyclodextrin and / or polysorbate 20. In some embodiments, the concentration of α-cyclodextrin in the reaction mixture is from about 10 mg / mL to about 40 mg / mL. In some embodiments, the concentration of α-cyclodextrin in the reaction mixture is from about 16 mg / mL to about 30 mg / mL, from about 15 mg / mL to about 20 mg / mL, or from about 10 mg / mL to about 15 mg / mL. In some embodiments, the concentration of α-cyclodextrin in the reaction mixture is about 20 mg / mL, about 17.5 mg / mL, or about 12.5 mg / mL. In some embodiments, the concentration of polysorbate 20 in the reaction mixture is from about 0.002% to about 0.05% (v / v). In some embodiments, the concentration of polysorbate 20 in the reaction mixture is from about 0.003% to about 0.03% (v / v). In some embodiments, the concentration of polysorbate 20 in the reaction mixture is about 0.0042% (v / v), about 0.0035% (v / v), about 0.0026% (v / v), or about 0.02% (v / v). In some embodiments, the reaction mixture comprises a detergent. In some embodiments, the detergent comprises sodium dodecyl sulfate. In some embodiments, the concentration of the detergent in the reaction mixture is from about 3 mg / mL to 300 mg / mL. In some embodiments, the concentration of the detergent in the reaction mixture is from about 10 mg / mL to 100 mg / mL. In some embodiments, the concentration of the detergent in the reaction mixture is about 33.3 mg / mL.

[0175] The present disclosure also provides a kit for carrying out the methods described herein. A kit according to the present disclosure comprises at least one or more of the following: oligonucleotides described herein for amplification of a target nucleic acid, and oligonucleotides (e.g., probes) described herein for determining the presence or absence of an amplification product of a target nucleic acid. In some embodiments, any combination of oligonucleotides described herein is present in the kit. The kit may further comprise a number of optional components such as, for example, capture probes, such as poly-(k) capture probes as described in US2013 / 0209992, and / or poly-(R) capture probes as described in US2020 / 0165599. Other reagents that may be present in the kit include, for example, reagents suitable for performing in vitro amplification such as buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP, and / or ATP, CTP, GTP, and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or an RNA polymerase). The oligonucleotides described herein may be packaged in a variety of different embodiments, and one of ordinary skill in the art will understand that the present disclosure encompasses many different kit configurations. For example, the kit may contain amplification oligonucleotides for only one, two, three, four, or all of Salmonella, C. jejuni, C. coli, Shigella, and STEC. Additionally, for kits that include detection probes along with an amplification oligomer combination, the amplification oligonucleotides and the detection probe oligonucleotides for the reaction mixture are bound by a common target region (i.e., the reaction mixture will include a probe that binds to a sequence that can be amplified by the amplification oligonucleotide combination of the reaction mixture). In certain embodiments, the kit further comprises a set of instructions for carrying out the methods according to the present disclosure, and the instructions may be associated with an accompanying document and / or the packaging of the kit or its components.

[0176] According to the present disclosure, there is also provided, for example, a method (e.g., a multiplex method) for determining the presence or absence of at least one enteric pathogen, including Salmonella, C. jejuni, C. coli, Shigella, and STEC in a sample, by using one or more of the oligonucleotides disclosed herein. Any of the methods disclosed herein should also be understood as a disclosure of the corresponding use of the materials involved in the method for the purpose of the method. Any of the oligonucleotides, and any combination (e.g., kits and compositions) containing such oligonucleotides, are also disclosed for use in detecting or quantifying at least one enteric pathogen and for use in the preparation of a composition for detecting or quantifying at least one enteric pathogen.

[0177] Broadly speaking, the method can include one or more of the following components: target capture, where target nucleic acid (from a sample, such as a clinical sample) anneals to a capture oligonucleotide; isolation, e.g., washing, to remove materials not associated with the capture oligonucleotide; amplification; and amplicon detection, e.g., amplicon quantification, which can be performed in real time along with the amplification. Certain embodiments involve each of the aforementioned steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two adjacent components listed above, e.g., washing and amplification, or amplification and detection.

[0178] In some embodiments, amplification comprises: (1) contacting a sample with at least two oligonucleotides for amplifying a target nucleic acid target region corresponding to a target nucleic acid, wherein the oligonucleotides comprise at least two amplification oligonucleotides as described above (e.g., one or more oriented in the sense direction and one or more oriented in the antisense direction for exponential amplification); (2) performing an in vitro nucleic acid amplification reaction, wherein any target nucleic acid present in the sample is used as a template for generating an amplification product; and (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of at least one enteric pathogen in the sample, including Salmonella, C. jejuni, C. coli, Shigella, and STEC, or quantifying the amount of the target nucleic acid in the sample.

[0179] The detection method according to the present disclosure can further comprise obtaining a sample for use in subsequent steps of the method. In certain embodiments, "obtaining" the sample used can include, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed and / or retrieving the sample from a location within the facility where one or more steps of the method are performed (e.g., from storage or other repository).

[0180] In certain embodiments, the method further comprises purifying the target nucleic acid from other components in the sample prior to amplification, such as, for example, prior to a capture step. Such purification can include methods for separating and / or concentrating organisms contained in the sample from other sample components or methods for removing or degrading non-nucleic acid sample components, such as proteins, carbohydrates, salts, lipids, etc. In some embodiments, the nucleic acid in the sample is degraded, for example, with DNase, and optionally the DNase is removed or inactivated or the degraded nucleic acid is removed.

[0181] In certain embodiments, purifying the target nucleic acid involves capturing the target nucleic acid and specifically or non-specifically separating the target nucleic acid from other sample components. Non-specific target capture methods can involve selective precipitation of nucleic acids from a substantially aqueous mixture, attachment of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating the nucleic acids from a mixture containing the target nucleic acid and other sample components.

[0182] Target capture typically occurs in a solution-phase mixture containing one or more capture probe oligonucleotides that hybridize to the target nucleic acid sequence under hybridization conditions. For embodiments that include a capture probe tail, the target:capture-probe complex is captured by adjusting the hybridization conditions such that the capture probe tail hybridizes to an immobilization probe. Certain embodiments use a particulate solid support such as paramagnetic beads.

[0183] Isolation can follow capture, and the complex on the solid support is separated from other sample components. Isolation can be achieved by any suitable technique, for example, washing the support associated with the target sequence one or more times (e.g., 2 or 3 times) to remove other sample components and / or unbound oligomers. In embodiments using a particulate solid support such as paramagnetic beads, the particles associated with the target are suspended in a wash solution and, in some embodiments, can be recovered from the wash solution by using magnetic attraction. To limit the number of handling steps, the target nucleic acid can be amplified by simply mixing the target sequence in the complex on the support with amplification oligomers and proceeding with the amplification step.

[0184] Exponentially amplifying the target array utilizes an in vitro amplification reaction that uses at least two amplification oligonucleotides adjacent to the target region to be amplified. In some embodiments, at least one oligonucleotide as described above is provided. In some embodiments, a plurality of oligonucleotide pairs are provided, the plurality including oligonucleotide pairs configured to hybridize to at least one, two, three, four, or all of Salmonella, C. jejuni, C. coli, Shigella, and STEC target nucleic acids. The amplification reaction can be cyclic or isothermal. Suitable amplification methods include, for example, replisome-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-related amplification (TMA).

[0185] The detection step can be performed using any of a variety of known techniques for detecting a signal specifically related to the amplified target sequence, such as, for example, hybridizing the amplification product with a labeled detection probe and detecting the signal resulting from the labeled probe (including in some embodiments from a label released from the probe after hybridization), performing electrophoresis on the sample and / or the amplification product, determining the sequence of the amplification product, etc. In some embodiments, the labeled probe includes a second moiety, such as a quencher or another moiety that interacts with the first label, as discussed above. The detection step can also provide additional information regarding the amplified sequence, such as, for example, all or a portion of its nucleobase sequence. Detection can be performed after the amplification reaction is complete or, for example, in real-time, simultaneously with amplifying the target region. In one embodiment, the detection step enables homogeneous detection, for example, detecting the hybridized probe without removing the unhybridized probe from the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174). In some embodiments, the nucleic acid is associated with a surface that results in a physical change, such as a detectable potential change. The amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acid or a dye associated therewith (e.g., an intercalating agent such as ethidium bromide or SYBR Green) or by detecting an increase in the dye associated with the nucleic acid in solution phase. Other detection methods can use nucleic acid detection probes configured to specifically hybridize to sequences in the amplified product and detect the presence of the probe:product complex or can be by using a complex of probes that can amplify a detectable signal associated with the amplified product (see, e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481, each of which is incorporated herein by reference). The directly or indirectly labeled probe specifically associated with the amplified product provides a detectable signal indicating the presence of the target nucleic acid in the sample.In particular, the amplified product contains a target sequence in or complementary to a target nucleic acid of at least one enteric pathogen, and the probe will bind directly or indirectly to the sequence contained in the amplified product to indicate the presence or absence of the pathogen in the tested sample.

[0186] In embodiments where the amplified product is detected near or at the end of the amplification step, a linear detection probe can be used to provide a signal indicative of hybridization of the probe to the amplified product. An example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is carried out by chemiluminescence using a luminometer (see, e.g., PCT Patent Application Publication No. 89 / 002476). In other embodiments using real-time detection, the detection probe can be a hairpin probe such as a molecular beacon, molecular torch, or hybridization switch probe that is labeled with a reporter moiety that is detected, for example, when the probe binds to the amplified product. Such probes can include a target hybridization sequence and a non-target hybridization sequence. Various forms of such probes are described, for example, in U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication Nos. 2006 / 0068417A1 and 2006 / 0194240A1.

[0187] In some embodiments, in the amplification reaction, the amplification oligonucleotide can preferentially hybridize to a target nucleic acid, such as a target sequence of a pathogen. In some embodiments, in the detection step, the probe can preferentially hybridize to a target nucleic acid, such as an amplification product. For example, under stringent hybridization conditions, the amplification or detection probe oligonucleotide can hybridize to its target nucleic acid to form a stable oligonucleotide:target hybrid, but does not form a sufficient number of stable oligonucleotide:non-target hybrids. In some embodiments, there is at least a 5-fold difference, at least a 10-fold difference, at least a 20-fold difference, at least a 50-fold difference, at least a 75-fold difference, at least a 100-fold difference, at least a 200-fold difference, at least a 500-fold difference, at least a 1,000-fold difference, or at least a 2,000-fold difference between the target and non-target hybridization signals in the test sample (i.e., the ratio of the signal intensities of the oligonucleotide:target hybrid and the oligonucleotide:non-target hybrid). In some embodiments, the non-target hybridization signal in the test sample is below the background signal level.

[0188] In some embodiments, the amplification reaction and / or detection step may be performed under stringent hybridization conditions. In some embodiments, such stringent hybridization conditions allow an oligonucleotide (e.g., an amplification oligonucleotide or a probe) to preferentially hybridize to a target nucleic acid (such as a target sequence of a pathogen or an amplification product) rather than to a nucleic acid derived from a closely related non-target nucleic acid. In different embodiments, the actual reaction environment that can be used for stringent hybridization may vary depending on the GC content and length of the oligonucleotide, the degree of similarity between the oligonucleotide sequence and the sequences of non-target nucleic acids that may be present in the test sample, and factors including the target sequence. In some embodiments, the hybridization conditions for the amplification reaction and / or detection step are such that when the monovalent salt, e.g., KCl, has a salt concentration of about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, or about 1.0 M, the temperature corresponds to about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, or about 65 °C.

[0189] For example, by using one or more of the oligonucleotides disclosed herein, a method (e.g., a multiplex method) for determining the presence or absence of at least one enteric pathogen, including Salmonella, C. jejuni, C. coli, Shigella, and STEC, in a sample may have a detection sensitivity of 25 - 500 CFU / mL, 50 - 500 CFU / mL, 75 - 500 CFU / mL, 100 - 500 CFU / mL, 25 - 300 CFU / mL, 50 - 300 CFU / mL, 75 - 300 CFU / mL, 100 - 300 CFU / mL, 25 - 150 CFU / mL, 50 - 150 CFU / mL, 75 - 150 CFU / mL, 100 - 150 CFU / mL, 25 - 100 CFU / mL, 50 - 100 CFU / mL, 75 - 100 CFU / mL, 25 - 75 CFU / mL, 50 - 75 CFU / mL, or 25 - 50 CFU / mL.

[0190] A method for synthesizing one or more (e.g., one or more pairs) of the oligonucleotides disclosed herein, wherein the oligonucleotide is useful for determining the presence or absence of at least one enteric pathogen, including Salmonella, C. jejuni, C. coli, Shigella, and STEC, is also provided. The method can include, for example, (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is attached (e.g., covalently) to the solid support at the 3'-position; (b) attaching the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 times, thereby generating at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleobase residues attached to the solid support; and (d) cleaving at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleobase residues generated in step (c), thereby obtaining an oligonucleotide or a plurality of oligonucleotides. In some embodiments, the oligonucleotide has a length of 18 to 32 consecutive nucleobase residues. In some embodiments, the oligonucleotide has a length of 20 to 30 consecutive nucleobase residues.

[0191] A method for synthesizing one or more of the oligonucleotides disclosed herein can be a solid-phase method. For example, phosphoramidite solid-phase chemistry for joining nucleotides by phosphodiester bonds is described in Caruthers et al., “Chemical Synthesis of Deoxynucleotides by the Phosphoramidite Method,” Methods Enzymol. 154:287 (1987). As another example, automated solid-phase chemical synthesis using cyanoethyl phosphoramidite precursors is described in Barone et al., “In Situ Activation of bis-dialkylaminephosphines - a New Method for Synthesizing Deoxyoligonucleotides on Polymer Supports,” Nucleic Acids Res. 12(10):4051 (1984). As another example, U.S. Patent No. 5,449,769, entitled “Method and Reagent for Sulfurization of Organophosphorous Compounds,” discloses procedures for synthesizing oligonucleotides containing phosphorothioate bonds. In addition, U.S. Patent No. 5,811,538, entitled “Process for the Purification of Oligomers,” discloses the synthesis of oligonucleotides having different linkages, including methylphosphonate linkages. Further, methods for the organic synthesis of oligonucleotides are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) Chapter 10.

[0192] After the synthesis and purification of specific oligonucleotides, several different procedures can be utilized to purify and control the quality of the oligonucleotides. Suitable procedures include electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography).

Example

[0193] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the present disclosure in any way.

[0194] Example 1 - Real-time PCR Amplification and Detection of GI-Bacterial Panel Targets Using Different Combinations of Primers and Probes Several primer and probe combinations for the real-time PCR amplification and detection of Salmonella, Campylobacter, Shigella / Enteroinvasive E.coli (Shigella / EIEC), and Shigatoxigenic E.coli (STEC) targets were tested.

[0195] The amplification and detection reactions were performed using a Panther Fusion instrument (Hologic, Inc. San Diego, CA). Typically, 20 μL of amplification reagent was combined in the reaction wells of a multiwell plate containing 5 μL of target nucleic acid. The multiwell plate was placed in the Panther Fusion instrument and subjected to thermal cycling. The real-time amplification and detection reactions were generally carried out by thermal cycling over 45 cycles (denaturation at 95 °C for 8 seconds and annealing and extension at 60 °C for 25 seconds), and fluorescence emission measurements were taken every 30 seconds. The fluorescence curve profiles for the target nucleic acid were evaluated for Ct and RFU signals.

[0196] Example 2 - Analytical Sensitivity - Bacterial Detection Limit An analytical sensitivity experiment was conducted to determine the limit of detection (LoD) of the target nucleic acids of six individual bacterial organisms using multiplexed primer-probe combinations. The primers and probes used in the experiment are shown in Table 1. The known stock concentrations of each organism (available from American Type Tissue Culture, Manassas, VA) were serially diluted in a fecal matrix to provide a series of five dilution concentrations for each organism. Salmonella typhimurium, Campylobacter coli, Campylobacter jejuni, Shigella sonnei, Shiga Toxin-producing Escherichia coli O157:H7 (STEC stx1 and STEC stx2) were serially diluted to provide the following concentrations: 1,000 CFU / mL, 500 CFU / mL, 300 CFU / mL, 150 CFU / mL, and 100 CFU / mL. Campylobacter jejuni was serially diluted to provide the following concentrations: 1,000 CFU / mL, 500 CFU / mL, 300 CFU / mL, 100 CFU / mL, and 75 CFU / mL. Each dilution was tested in quintuplicate, and the Ct and RFU data are shown in Table 2. The lowest concentration that was 100% positive for each organism was then further tested in 20 replicates (dilutions made in both fecal matrix and Cary-Blair fecal matrix), and the Ct and RFU data are shown in Table 3. Sample processing, target capture, amplification, and detection reactions were generally performed as described herein. Internal control nucleic acids were present in each amplification reaction.

[0197]

Table 1

[0198]

Table 2

[0199]

Table 3

[0200] Data from this experiment showed 100% positivity (5 / 5) at as low as 100 CFU / mL for Salmonella typhimurium, Shigella sonnei, STEC stx1, and STEC stx2. Campylobacter coli was 100% positive at 300 CFU / mL and 80% positive (4 / 5) at 100 CFU / mL, and Campylobacter jejuni was 100% positive at 75 CFU / mL. The confirmation test showed 100% positivity (20 / 20) for each of Campylobacter coli, Campylobacter jejuni, Shigella sonnei, STEC stx1, and STEC stx2 in two different fecal matrices. Salmonella typhimurium was 90% positive in the raw fecal matrix and 95% positive in the Cary - Blair fecal matrix. These data indicate that the multiplex primer / probe combination has a sensitivity of 75 - 150 CFU / mL.

[0201] Example 3 - Primer - Probe Combinations for Amplification and Detection of Salmonella Several primer and probe combinations were prepared and tested for the amplification and detection of Salmonella enterica. The combinations are listed in Table 4 below. These primer - probe combinations were tested against three concentrations of serially diluted stock concentrations of Salmonella enterica. The diluents were used as sample transport reagents, and concentrations of 500 CFU / mL, 150 CFU / mL, and 50 CFU / mL were tested. The negative reaction wells were the sample transport medium. The reaction conditions were set in three ways, and real - time amplification and detection reactions were carried out. The RFU and Ct results are shown in Table 5.

[0202]

Table 4 - 1

Table 4 - 2

[0203]

Table 5

[0204] Primer and probe combinations 1 and 2 showed only negative results in this experiment and thus the results are not shown in Table 5. Each of combinations 3 - 7 showed robust RFU relative to the background. Combinations 4 and 5 showed the most robust RFU and the fastest Ct in this experiment.

[0205] Example 4 - Analytical Specificity and Interference The analytical specificity was tested for an exemplary multiplex assay for detecting Salmonella, Shigella, Campylobacter (C. jejuni and C. coli, undifferentiated), and STEC (stx - 1 and stx - 2 undifferentiated). The assay was run as a real - time, multiplex PCR reaction utilizing the primer and probe combinations of Table 1 and the cycling parameters.

[0206] The analytical specificity of the PCR assay was determined using a total of 81 organisms divided into 17 panels listed in Tables 6 and 6.1. These 81 organisms are commonly found in fecal samples (organisms available from American Type Tissue Culture, Manassas, VA). The 17 organism panels were spiked into a negative Cary - Blair fecal matrix. The concentration of the organisms was 1E6 CFU / mL in each test, except for Bifidobacterium adolescentis, Egglerthella lenta, and Peptostreptococcus micros which had a concentration of 1E6 rRNA copies / mL, and Entamoeba histolytica which had a concentration of 1E4 cells / mL. The positive controls were Salmonella enterica (strain V1796), Campylobacter coli (strain RO 268), Shigella flexneri (strain 24570), and STEC (strain CDC 1999 - 3302) spiked into the Cary - Blair fecal matrix at 3 times the LoD. The negative control was the Cary - Blair fecal matrix only. The reactions were run in triplicate. Reactivity or positivity was defined as an amplification curve exceeding the Ct threshold for each channel. Any curve below this threshold was considered negative or non - reactive. The Ct thresholds used were: FAM, 600; HEX, 1000; ROX, 500; Q670, 600; and Q705, 1000. The results are shown in Tables 7, 7.1, 8, and 8.1.

[0207]

Table 6 - 1

Table 6 - 2

[0208]

Table 6.1

[0209]

Table 7

[0210]

Table 7.1

[0211]

Table 8

[0212]

Table 8.1

[0213] As shown in Table 7, the primers and probes did not react with any of the challenge organisms listed in Table 6. Also, as shown in Table 8, when the target bacteria were present in the reaction wells, the primers and probes showed 100% positivity. Therefore, these primers and probes do not demonstrate cross-reactivity with organisms commonly found in feces or organisms that cause disease states similar to the target organisms. The primers and probes showed high specificity even in the presence of commonly found non-target organisms.

[0214] In addition, since Shigella and EIEC have substantially the same ipaH gene, the Shigella primers and probes reacted with the organisms in Table 6.1 as shown in Table 7.1. Also, as shown in Table 8.1, when the target bacteria were present in the reaction wells, the primers and probes also showed 100% positivity. As such, the Shigella primers and probes can be used to detect the presence of EIEC in a sample.

[0215] Example 5 - Shigella Primer and Probe Screening Several primer and probe combinations were prepared and tested for the amplification and detection of Shigella sonnei. The combinations are listed in Table 9 below. These primer-probe combinations were tested against three serial dilution levels of a stock concentration of Shigella sonnei. The diluents were used as sample transport reagents, and concentrations of 500 CFU / mL, 150 CFU / mL, and 50 CFU / mL were tested. Negative reaction wells were the sample transport medium. The reaction conditions were set in three ways, and real-time amplification and detection reactions were carried out. The RFU and Ct results were analyzed and are shown below.

[0216] [Table 9]

[0217] The results showed 100% positivity (Ct threshold set at 1,000 RFU) for each primer-probe combination. Primer-probe combination 2 showed the highest RFU (approximately 12,000 RFU at 500 cfu / mL), but at 50 cfu / mL, one of the replicates dropped to approximately 2,000 RFU, while the other two replicates were approximately 8,000 RFU. Primer-probe combination 1.2 showed good overall results with consistent RFU and Ct values for the tested reaction conditions. The RFU and Ct results are shown in Table 10.

[0218] [Table 10-1] [Table 10-2]

[0219] Example 6 - Coinfection / Competitive Interference In a multiplex PCR assay, the performance of detecting two or more target organisms in a single sample was measured using the primer-probe combinations shown in Table 1. To create co-infected samples, a negative Cary-Blair stool matrix was spiked with combinations of target organisms at high concentration (1E6 CFU / mL) and low concentration (3xLoD) as shown in Table 11. The primer-probe combinations were tested against low concentrations of target organisms in the absence of co-infecting substances and further tested with combinations of low concentrations of target organisms and high concentrations of separate target organisms (see Table 12).

[0220]

Table 11

[0221]

Table 12

[0222] Detection of both high and low concentrations of all target bacteria was 100%. There were no false positives and no competitive interference was observed. The data indicate that the primer-probe combinations can accurately detect multiple target bacteria present in a single sample using multiplex PCR.

[0223] Example 7 - Reduction of Inhibition of Amplification Reactions by α-Cyclodextrin and / or Polysorbate 20 For example, detergents present in wash buffers are known to inhibit or reduce nucleic acid amplification reactions. To investigate the ability of α-cyclodextrin and polysorbate 20 to mitigate such negative / inhibitory effects, several PCR reaction mixtures were prepared for testing with samples containing detergents.

[0224] A master mix was prepared to contain 0.46 U / μL of DNA polymerase, 0.5 U / μL of reverse transcriptase, 0.2 U / μL of RNase inhibitor, 0.25 mM each of dNTPs, 0.05 mM of dUTP, inorganic salts including 81 and 5.1 mM of KCl and MgCl2, 0.1 mM of EDTA, and primers and probes for amplification and detection of the target nucleic acid. The master mix was separated into conditions (A) - (F), conditions (B) and (D) further contained 0.025% (v / v) of polysorbate 20, conditions (B), (C), and (F) further contained 12.5 mg / mL of α - cyclodextrin, condition (C) further contained 0.13% (v / v) of polysorbate 20, and condition (E) further contained 50 mg / mL of α - cyclodextrin. Samples were prepared by spiking the target nucleic acid into a suitable medium and incubating in a buffer reagent containing poly - T - coated magnetic microparticles and target - capture oligomers to bind the target nucleic acid to the magnetic solid support. The magnetic microparticles and bound nucleic acid were separated, and after additional separation and washing, an eluate containing the nucleic acid was recovered. The eluate was divided into separate containers, and one of the eluate portions was further spiked with 30% (v / v) of wash buffer to produce an eluate containing 100 mg / mL of sodium dodecyl sulfate to simulate carry - over of wash buffer from the upstream sample processing step. Each of the master mix conditions (A) - (F) was combined with an aliquot of either wash buffer - spiked or non - spiked eluate (20 μL of master mix, 10 μL of eluate, 30 μL total reaction volume per replicate). Thus, the final concentration of polysorbate 20 and / or α - cyclodextrin in each reaction mixture was 2 / 3 of the concentration listed above for the master mix. Real - time amplification and detection reactions were set up for replicates and the reactions were performed using a thermal cycler (Panther Fusion Instrument, Hologic, Inc., San Diego, CA). The results are shown in Table 13 below.

[0225]

Table 13

[0226] The addition of α-cyclodextrin and polysorbate 20 to the reaction mixture reduced the negative / inhibitory effect of the detergent on the nucleic acid amplification reaction. The Ct and RFU values further showed that the combination of these two reagents had a synergistic reduction effect on detergent-induced inhibition compared to the reduction effect observed when either α-cyclodextrin or polysorbate 20 was used alone.

[0227]

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Claims

1. A set of oligonucleotides for determining the presence or absence of at least one enteric pathogen, comprising at least (a): (a) (i) A C. coli-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10 A set of oligonucleotides comprising the same.

2. (b) and (c): (b) (i) A Shigella-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, (ii) SEQ ID NO: 34 and SEQ ID NO: 22, or (iii) SEQ ID NO: 32 and SEQ ID NO: 33, and (c) (i) A Shiga toxigenic E. coli (STEC)-specific amplification oligonucleotide set comprising a first and a second oligomer each containing a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 3, (ii) SEQ ID NO: 49 and SEQ ID NO: 3, or (iii) SEQ ID NO: 4 and SEQ ID NO: 7 The set of oligonucleotides according to claim 1, further comprising at least one of the above.

3. The set of oligonucleotides according to claim 2, comprising the C. coli-specific amplification oligonucleotide set and at least one of the Shigella-specific amplification oligonucleotide set and the STEC-specific amplification oligonucleotide set.

4. The set of oligonucleotides according to claim 2, comprising the C. coli-specific amplification oligonucleotide set, the Shigella-specific amplification oligonucleotide set, and the STEC-specific amplification oligonucleotide set.

5. The Shigella-specific amplification oligonucleotide set comprises a first and a second oligomer each containing a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, and / or The STEC-specific amplification oligonucleotide set comprises a first and a second oligomer each containing a target hybridization sequence consisting of the nucleotide sequences of (i) SEQ ID NO: 20 and SEQ ID NO: 3 or (ii) SEQ ID NO: 4 and SEQ ID NO:

7. The set of oligonucleotides according to claim 4.

6. A C. coli detection probe comprising a target hybridization sequence consisting of the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 37, or SEQ ID NO: 39, (b) When the Shigella-specific amplification oligomer set includes first and second oligomers each comprising a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 16, When the Shigella-specific amplification oligomer set includes first and second oligomers each comprising a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 34 and SEQ ID NO: 22, SEQ ID NO: 46 or SEQ ID NO: 29, or When the Shigella-specific amplification oligomer set includes first and second oligomers each comprising a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 24 A Shigella detection probe comprising a target hybridization sequence consisting of the nucleotide sequence of, and / or (c) When the STEC-specific amplification oligomer set includes first and second oligomers each comprising a target hybridization sequence consisting of the nucleotide sequence of SEQ ID NO: 20 and SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 49 and SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 48, or When the STEC-specific amplification oligomer set includes first and second oligomers each comprising a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 7, a STEC detection probe comprising a target hybridization sequence consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 The set of oligonucleotides according to claim 4, further comprising.

7. The set of oligonucleotides according to claim 6, wherein one or more of the detection probes, or each, comprises a fluorescent dye compound.

8. The set of oligonucleotides according to claim 7, wherein each of the detection probes comprising the fluorescent dye compound further comprises a non-fluorescent quenching dye compound.

9. An oligonucleotide for determining the presence or absence of C. coli, wherein the oligonucleotide is SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 contains 5-methylcytosine), SEQ ID NO: 37 (where each of nucleotides 17, 22, 23, and 27 contains 5-methylcytosine), and SEQ ID NO: 39 (where each of nucleotides 2, 9, 12, 19, 22, 24, and 25 contains 5-methylcytosine), an oligonucleotide comprising any one of the sequences. **Claim 10** A kit comprising a set of oligonucleotides according to any one of claims 1 to 8 or an oligonucleotide according to claim 9. **Claim 11** A reaction mixture comprising a set of oligonucleotides according to any one of claims 1 to 8 or an oligonucleotide according to claim 9. **Claim 12** A method for determining the presence or absence of C. coli in a sample, the method comprising: (1) contacting a sample suspected of containing C. coli with a set of C. coli-specific amplification oligomers for amplifying a target region of a C. coli target nucleic acid, wherein the C. coli-specific amplification oligomer set comprises a first and a second oligomer each comprising a target hybridization sequence consisting of the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10; (2) performing an in vitro nucleic acid amplification reaction, which, if any C. coli target nucleic acid is present in the sample, is performed using the sample as a template for generating an amplification product corresponding to the C. coli target region; (3) detecting the presence or absence of the amplification product, thereby determining the presence or absence of C. coli in the sample. **Claim 13** A multiplex method for determining the presence or absence of C. coli and at least one of Shigella and Shiga-toxigenic E. coli (STEC) in a sample, the method comprising: (1) contacting a sample suspected of containing C. coli and at least one of Shigella and STEC with a set of oligonucleotides for amplifying a target region of a C. coli target nucleic acid and a target region of at least one of a Shigella target nucleic acid and an STEC target nucleic acid, wherein the set of oligonucleotides is the set of oligonucleotides according to any one of claims 2 to 5; (2) performing an in vitro nucleic acid amplification reaction, wherein any C. coli, Shigella, and STEC target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the C. coli, Shigella, and STEC target regions; (3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of C. coli and at least one of Shigella and STEC in the sample, the multiplexing method comprising:

14. (3) is contacting the sample with at least one of a C. coli detection probe, a Shigella detection probe, and an STEC detection probe, each of the detection probes being as defined in claim 6; performing electrophoresis on the sample, or determining the sequence of the one or more amplification products, if present, the multiplexing method according to claim 13 comprising:

15. A method for synthesizing an oligonucleotide, comprising: (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position; (b) bonding the 5'-position of the nucleobase residue furthest from the solid support to the 3'-position of another nucleobase residue; (c) repeating step (b) at least 13 additional times, thereby generating at least 15 contiguous nucleobase residues bound to the solid support; (d) cleaving the at least 15 contiguous nucleobase residues generated in step (c) to obtain the oligonucleotide, wherein the oligonucleotide comprises any one of the sequences of SEQ ID NO: 9 (each of nucleotides 2, 6, 13, 16, 23, 26, 28, and 29 comprises 5-methylcytosine), SEQ ID NO: 37 (each of nucleotides 17, 22, 23, and 27 comprises 5-methylcytosine), and SEQ ID NO: 39 (each of nucleotides 2, 9, 12, 19, 22, 24, and 25 comprises 5-methylcytosine) the method comprising:

16. A method for synthesizing a pair of oligonucleotide primers, comprising synthesizing a first oligonucleotide primer and synthesizing a second oligonucleotide primer, wherein each of synthesizing the first oligonucleotide primer and synthesizing the second oligonucleotide primer comprises (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bonded to the solid support at the 3'-position, (b) bonding the 5'-position of the nucleobase residue farthest from the solid support to the 3'-position of another nucleobase residue, (c) repeating step (b) at least 13 additional times, thereby generating at least 15 consecutive nucleobase residues bonded to the solid support, (d) cleaving the at least 15 consecutive nucleobase residues generated in step (c), thereby obtaining the oligonucleotide primer, and the first oligonucleotide primer and the second oligonucleotide primer each comprise the sequences of SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

Images