Methods, oligonucleotides and kits for detecting Streptococcus

Abstract

The invention discloses oligonucleotides, a kit and a method for detecting streptococci, which all involve a first primer pair and a first probe for detecting a conserved region in a genome of the genus Streptococcus; a second primer pair and a second probe for detecting a first conserved region in a genome of group B streptococci; a third primer pair and a third probe for detecting a second conserved region in the genome of group B streptococci that is different from the first conserved region. The fluorescent PCR technology is used to simultaneously detect the genus Streptococcus and its member group B streptococci, and when detecting group B streptococci, two different conserved regions in the genome of group B streptococci are amplified simultaneously to avoid missed detection.

Description

Technical Field

[0001] The invention belongs to the field of biological science and biotechnology, and particularly relates to a method, oligonucleotide and kit for detecting Streptococcus and group B Streptococcus. The fluorescent PCR technology is used to perform multiple detections on group B Streptococcus that may exist in clinical samples. Background Art

[0002] Streptococcus is a common gram-positive coccus in the pyogenic cocci. There are currently 69 species and subspecies of Streptococcus, which are widely distributed in nature, human and animal feces, and the nasopharynx of healthy people. Most of them are not pathogenic. The diseases caused by Streptococcus in the human body mainly include various suppurative inflammations, scarlet fever, neonatal sepsis, bacterial endocarditis, and hypersensitivity diseases such as rheumatic fever and glomerulonephritis.

[0003] According to the hemolytic characteristics on blood agar culture medium, Streptococcus can be divided into three different types: (1) Type alpha hemolytic streptococci, also known as grass-green streptococci, have a grass-green hemolytic ring around the colonies. They usually live in the human oropharynx, respiratory tract and intestines. They have weak pathogenicity and are mostly conditional pathogens. (2) Type beta hemolytic streptococci produce strong hemolytic toxins. On blood agar culture medium, they can cause a 2-4 mm wide, clearly defined, colorless and transparent hemolytic ring to appear around the colonies. They have strong pathogenicity and can cause humans and animals to suffer from a variety of diseases. (3) Type gamma streptococci are non-hemolytic. There is no hemolytic ring around the colonies and they are generally non-pathogenic.

[0004] According to the different antigenic structures of streptococci, they are divided into 20 groups, such as A, B, C, and D. In each group, there are several subgroups due to the differences in surface protein antigens. About 90% of streptococcal strains that are pathogenic to humans belong to group A, and groups B, C, D, and G are occasionally found.

[0005] Diseases caused by streptococci are complex and diverse. On the one hand, there are many types of bacteria, and they are both invasive and toxin-producing. On the other hand, all tissues and organs of the human body are highly susceptible, and allergic mechanisms are involved in the onset of disease. The invasive enzymes produced by streptococci include hyaluronidase, streptokinase, and streptodornase, etc.; the toxins produced include streptolysin and erythrotoxin. Allergic diseases such as rheumatic fever and acute glomerulonephritis can all be caused by group A streptococcal infection.

[0006] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a common Gram-positive streptococcus. It is named after the polysaccharide C substance in its cell wall, which belongs to Group B in the Lancefield antigen structure classification. According to the different antigenicity of its capsular polysaccharide, group B streptococcus is divided into 9 serotypes (Ⅰa, Ⅰb, Ⅱ~Ⅷ types). Group B streptococcus often exists in the rectum and vagina, and can cause maternal amniotic cavity infection, premature rupture of membranes, premature fetal birth, miscarriage, etc. According to statistics, about 10% to 30% of pregnant women are infected with group B streptococcus, of which 40% to 70% will be transmitted to newborns during delivery. If newborns carry this bacteria, about 1% to 3% will have early invasive infection, meningitis, pneumonia, sepsis, and 5% will die. Related studies have shown that the targeted use of antimicrobial drugs can effectively prevent the occurrence of adverse pregnancy outcomes such as premature rupture of membranes in parturients.

[0007] Given that group B streptococcal infection can cause adverse symptoms in pregnant women and newborns, prenatal screening for group B streptococci is extremely important. In 2010, the U.S. Centers for Disease Control and Prevention issued the "Guidelines for the Prevention of Perinatal Group B Streptococci," which recommends that pregnant women undergo group B streptococcal screening between 35 and 37 weeks of pregnancy.

[0008] For a long time, the diagnosis of GBS was mainly achieved by culturing rectal or vaginal swabs of pregnant women, blood or cerebrospinal fluid of newborns, but the false negative rate of conventional culture methods in detecting group B streptococcal infection in pregnant women can be as high as 50%. The application of selective culture medium can inhibit the growth of other microorganisms, and this selective culture has always been the gold standard for determining group B streptococcal infection. However, it takes 72 hours for selective culture to produce a negative result, and at least 48 hours for a positive result, so a method for rapid detection of group B streptococcus is needed to make up for this shortcoming.

[0009] Due to the implementation of antibiotic prevention strategies for group B streptococci and the widespread use of antibiotics, traditional culture and serological detection methods are inevitably insufficiently sensitive for patients with mild infections and those treated with antibiotics, and false negatives occur from time to time. The emergence and rapid development of molecular biology technology has provided a new technical platform for the rapid and sensitive diagnosis of group B streptococci. The following methods are mainly used for clinical diagnosis and (or) strain research of group B streptococci: molecular hybridization diagnosis, polymerase chain reaction (PCR) and related technical diagnosis, and molecular biology typing technology.

[0010] Molecular hybridization diagnosis is achieved by detecting the DNA or RNA of group B streptococci through specific probes. Commonly used techniques include in situ hybridization, Southern blot, and dot blot. Foreign countries often use hybridization kits designed for rRNA of group B streptococci to detect group B streptococcal infection. The sensitivity of detection in pregnant women is 94.17% to 100%, and the specificity is 96.19% to 99.15%. Recently, the use of fluorescent in situ hybridization (FISH) to detect the group B streptococcal carrier rate in pregnant women was reported. It was found that FISH can diagnose 98.13% of carriers, while the standard culture method has only a 64.14% positive rate. The results of FISH detection are relatively intuitive, but the test process is cumbersome, involving a wide variety of reagents, time-consuming and labor-intensive, and the results need to be interpreted by experienced professionals, and the interpretation of the results is highly subjective.

[0011] The development of polymerase chain reaction (PCR) technology has provided a powerful tool for rapid, sensitive and specific detection of pathogens in various clinical samples. Nested PCR detection of ribosomal operon genes is also a research direction in recent years. This method detects group B streptococci by amplifying the ribosomal 16S rRNA gene or the 16S-23S spacer. Its specificity is 87% to 96%, but there are certain false positives, which may be due to the high homology of ribosomal operons between bacterial species, such as 70% homology between 16S-23S rDNA of Streptococcus difficile and group B streptococci. The determination of the whole genome sequence of group B streptococci provides more information for the selection of other specific detection targets in addition to the ribosomal operon in group B streptococci. Some scholars have designed primers for the scpB gene encoding C5a peptidase to detect group B streptococci. Others have designed primers for the bca gene encoding Cα protein, the bac gene encoding Cβ protein, the alp2 and alp3 genes encoding Cα-like proteins, and the rib gene encoding rib protein to study the genetic distribution and variation of group B streptococcal surface proteins.

[0012] However, the existing technology does not detect Streptococcus at the same time as Group B Streptococcus, so that even if Group B Streptococcus is not present in the clinical sample, it can indicate the presence of other Streptococci, which helps clinicians to conduct further pathogen screening. In addition, the existing PCR technology often detects a single gene fragment of Group B Streptococcus, which is prone to missed detection. Summary of the invention

[0013] In view of the shortcomings of the prior art, the present invention provides a method, oligonucleotide and kit for detecting streptococci, selects two different conserved regions in the genome of group B streptococci (for example, one conserved region is from the Sip gene and the other conserved region is from the Cfb gene) to design two sets of primers and probes, and at the same time designs a set of primers and probes as an internal control according to a conserved sequence in the genome of the genus Streptococcus (for example, a conserved sequence from the 16S rRNA gene, the 23S rRNA gene or the 16S-23S rDNA spacer), and further introduces the human genome housekeeping gene β-globin as an internal reference to establish a double quality control and double target verification system, which can effectively avoid a series of abnormal results encountered in the detection of clinical samples, and can also solve the problem of missed detection of group B streptococci.

[0014] In the present invention, x1, x2, x3 and x4 located at the 5' end of each probe are fluorescent reporter groups, and y1, y2, y3 and y4 located at the 5' end of each probe are quencher groups that emit fluorescence or do not emit fluorescence.

[0015] The fluorescent reporter gene and quencher group of the same probe in the present invention are given in the form of "FAM-TARMA / BHQ1" or "VIC / HEX / JOE-TAMRA / BHQ1", the left side of "-" represents the fluorescent reporter group, and the right side of "-" represents the quencher group. FAM-TARMA / BHQ1 means that the fluorescent reporter gene of a probe is FAM, and the quencher group can be selected from TARMA or BHQ1. VIC / HEX / JOE-TAMRA / BHQ1 means that the fluorescent reporter gene of a probe can be selected from VIC, HEX or JOE, and the quencher group can be selected from TAMRA or BHQ1.

[0016] The present invention provides an oligonucleotide for detecting streptococci, characterized in that the oligonucleotide comprises (1) a first primer pair and a first probe for detecting a conserved region in the genome of the genus Streptococcus; (2) a second primer pair and a second probe for detecting a first conserved region in the genome of group B streptococci; and (3) a third primer pair and a third probe for detecting a second conserved region in the genome of group B streptococci that is different from the first conserved region.

[0017] Furthermore, the oligonucleotide also includes (4) a fourth primer pair and a fourth probe for detecting an internal standard gene.

[0018] Furthermore, the conserved region of the Streptococcus genome is from the 16S rRNA gene, the 23S rRNA gene or the 16S-23S rDNA spacer.

[0019] Furthermore, the first conserved region is from the Sip or Cfb gene.

[0020] Furthermore, the second conserved region is from the Sip or Cfb gene.

[0021] Furthermore, the base sequences of the first primer pair and the first probe are SEQ ID NOs: 1 to 3, respectively.

[0022] Furthermore, the base sequences of the second primer pair and the second probe are SEQ ID NOs: 4 to 6, respectively.

[0023] Furthermore, the base sequences of the third primer pair and the third probe are SEQ ID NOs: 7 to 9, respectively.

[0024] Furthermore, the base sequences of the fourth primer pair and the fourth probe are SEQ ID NOs: 10 to 12, respectively.

[0025] Further, the fluorescent reporter gene and quencher group of each probe are selected from any one of FAM-TARMA / BHQ1, VIC / HEX / JOE-TAMRA / BHQ1, ROX / Texas Red-BHQ2 and Cy5 / LC Red640-BHQ2 / BHQ3.

[0026] The present invention also provides a method for detecting streptococci, the method comprising: (1) extracting DNA from a sample; (2) performing fluorescent PCR amplification on the DNA in (1) in a single tube in the presence of a set of primers and probes; (3) determining whether group B streptococci are present in the sample, and if not, determining whether other streptococcal bacteria are present, the set of primers and probes comprising a first primer pair and a first probe for detecting a conserved region in the genome of the streptococcus genus; a second primer pair and a second probe for detecting a first conserved region in the genome of the group B streptococci; and a third primer pair and a third probe for detecting a second conserved region in the genome of the group B streptococci that is different from the first conserved region.

[0027] Furthermore, the set of primers and probes also includes a fourth primer pair and a fourth probe for detecting an internal standard gene.

[0028] Furthermore, the conserved region of the Streptococcus genome is from the 16S rRNA gene, the 23S rRNA gene or the 16S-23S rDNA spacer.

[0029] Furthermore, the first conserved region is from the Sip or Cfb gene.

[0030] Furthermore, the second conserved region is from the Sip or Cfb gene.

[0031] Furthermore, the base sequences of the first primer pair and the first probe are SEQ ID NOs: 1 to 3, respectively.

[0032] Furthermore, the base sequences of the second primer pair and the second probe are SEQ ID NOs: 4 to 6, respectively.

[0033] Furthermore, the base sequences of the third primer pair and the third probe are SEQ ID NOs: 7 to 9, respectively.

[0034] Furthermore, the base sequences of the fourth primer pair and the fourth probe are SEQ ID NOs: 10 to 12, respectively.

[0035] Further, the fluorescent reporter gene and quencher group of each probe are selected from any one of FAM-TARMA / BHQ1, VIC / HEX / JOE-TAMRA / BHQ1, ROX / Texas Red-BHQ2 and Cy5 / LC Red640-BHQ2 / BHQ3.

[0036] The present invention also provides a kit for detecting streptococci, the kit comprising a fluorescent PCR reaction solution, the fluorescent PCR reaction solution comprising oligonucleotides, the oligonucleotides comprising (1) a first primer pair and a first probe for detecting a conserved region in a Streptococcus genome; (2) a second primer pair and a second probe for detecting a first conserved region in a group B streptococcus genome; and (3) a third primer pair and a third probe for detecting a second conserved region in a group B streptococcus genome that is different from the first conserved region.

[0037] Furthermore, the oligonucleotide comprises (4) a fourth primer pair and a fourth probe for detecting an internal standard gene.

[0038] Furthermore, the conserved region of the Streptococcus genome is from the 16S rRNA gene, the 23S rRNA gene or the 16S-23S rDNA spacer.

[0039] Furthermore, the first conserved region is from the Sip or Cfb gene.

[0040] Furthermore, the second conserved region is from the Sip or Cfb gene.

[0041] Furthermore, the base sequences of the first primer pair and the first probe are SEQ ID NOs: 1 to 3, respectively.

[0042] Furthermore, the base sequences of the second primer pair and the second probe are SEQ ID NOs: 4 to 6, respectively.

[0043] Furthermore, the base sequences of the third primer pair and the third probe are SEQ ID NOs: 7 to 9, respectively.

[0044] Furthermore, the base sequences of the fourth primer pair and the fourth probe are SEQ ID NOs: 10 to 12, respectively.

[0045] Further, the fluorescent reporter gene and quencher group of each probe are selected from any one of FAM-TARMA / BHQ1, VIC / HEX / JOE-TAMRA / BHQ1, ROX / Texas Red-BHQ2 and Cy5 / LC Red640-BHQ2 / BHQ3.

[0046] Furthermore, the kit further comprises a nucleic acid extracting solution 1 and a nucleic acid extracting solution 2, wherein the nucleic acid extracting solution 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1%-2% SDS, and the nucleic acid extracting solution 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1%-2% mercaptoethanol and 1%-5% Chelex-100.

[0047] The present invention also provides a kit for detecting streptococci, the kit comprising a PCR reaction solution, an enzyme mixture, a positive control, a negative control and a nucleic acid extract, including: (1) primers and probes for detecting streptococci, whose base sequences are SEQ ID NOs: 1 to 3; (2) primers and probes for detecting the Sip gene of group B streptococci, whose base sequences are SEQ ID NOs: 4 to 6; (3) primers and probes for detecting the Sip gene of group B streptococci, whose base sequences are SEQ ID NOs: 7 to 9; (4) primers and probes for detecting internal reference genes, whose base sequences are SEQ ID NOs: 10 to 12.

[0048] Furthermore, the concentration of each primer and probe ranges from 75 to 300 nM.

[0049] Furthermore, the nucleic acid extract comprises nucleic acid extract 1 and nucleic acid extract 2, wherein nucleic acid extract 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1%-2% SDS; nucleic acid extract 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100.

[0050] Furthermore, the PCR reaction solution also includes 10-50 mM Tris (pH 8.0-9.2), 10-50 mM KCl, 10-20 mM ammonium sulfate, 0.01%-0.1% Tween20, 0.2-2 mg / mL BSA, 0.1-0.3 mM dATP, 0.1-0.3 mM dTTP, 0.1-0.3 mM dCTP, 0.1-0.3 mM dGTP, 0.1-0.3 mM dUTP, and 3-6 mM magnesium chloride.

[0051] Furthermore, the operating procedures of the kit are as follows: (1) extracting sample DNA by two-step method: in the first step, taking 1 mL of sample and centrifuging at 12000 rpm, discarding the supernatant, adding 0.5 mL of nucleic acid extracting solution 1, vortexing to mix, and discarding the supernatant; in the second step, adding 100 μL of nucleic acid extracting solution 2, boiling at 100° C. for 10 min, centrifuging at 12000 rpm, and keeping the supernatant nucleic acid for later use; (2) preparing PCR reagents: mixing the PCR reaction solution and the enzyme mixture, and adding the sample DNA extracted in (1) and the negative quality control and positive quality control, respectively; (3) fluorescent PCR detection: performing fluorescent PCR amplification, and determining whether Streptococcus and its member group B Streptococcus are present in the sample after the amplification is completed.

[0052] Beneficial effects: (1) The present invention selects two different conserved regions in the genome of group B streptococci (for example, one conserved region is from the Sip gene and the other conserved region is from the Cfb gene) to design two sets of primers and probes, and at the same time designs a set of primers and probes based on a conserved sequence in the genome of the genus Streptococcus (for example, a conserved sequence from the 16S rRNA gene, the 23S rRNA gene or the 16S-23S rDNA spacer) as an internal control, and then introduces the human genome housekeeping gene β-globin as an internal reference to establish a double quality control and double target verification system, thereby effectively avoiding a series of factors such as clinical sample sampling errors and nucleic acid extraction errors that cause abnormal results. More importantly, this can also solve the problem of missed detection of group B streptococci; (2) Given that group B streptococci are also a member of the genus Streptococcus, when group B streptococci are present in clinical samples, when the conserved regions of the Sip and Cfb genes to be detected have not mutated, the 16S rRNA, Sip and Cfb genes should all show obvious S curves when detected by fluorescent PCR. Therefore, there are multiple indicators to determine whether group B streptococci exist in clinical samples, which can further reduce the occurrence of false positives. (3) In view of the different evolutionary pressures and mutation rates of group B streptococci in different human environments, it is possible that group B streptococci exist in clinical samples, but are not detected due to mutations in the gene to be detected (Sip is used as an example). The present invention can also determine whether group B streptococci exist based on whether obvious S curves appear in fluorescent PCR detection of 16S rRNA and Cfb genes, thereby minimizing the missed detection of group B streptococci due to gene sequence mutations. (4) When group B streptococci do not exist in clinical samples, no obvious S curves will appear in fluorescent PCR detection of Sip and Cfb genes. In this case, if no obvious S curves appear in fluorescent PCR detection of 16S rRNA gene, this indicates that streptococci do not exist in clinical samples. If the 16S rRNA gene is detected, the S curves will not appear. When the rRNA gene is tested by fluorescent PCR, an obvious S curve appears, indicating that there are streptococci other than group B streptococci in the clinical sample, which helps clinicians to conduct further pathogen screening; (5) The present invention can detect group B streptococci from complex samples, has the advantages of high sensitivity, good specificity, rapid and objective detection results, etc., and provides reliable results for diagnosing infections of Streptococcus and its members group B streptococci. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : The detection results of nucleic acids extracted by the alkaline lysis method of the present invention.

[0054] Figure 2 : Detection results of nucleic acid extracted by magnetic bead method as a control.

[0055] Figure 3 : The test result of the present invention to positive reference substance P1.

[0056] Figure 4 : The present invention is to the detection result of positive reference substance P2.

[0057] Figure 5 : The present invention is to the detection result of positive reference substance P3.

[0058] Figure 6 : The present invention is to the detection result of positive reference substance P4.

[0059] Figure 7 : The present invention is to the detection result of positive reference substance P5.

[0060] Figure 8 : The present invention is directed to the test results of negative reference products N1-N10.

[0061] Fig. 9 : The preliminary judgment result diagram of the sensitivity of Cfb gene detection in Cy5 channel of the present invention.

[0062] Fig.10 : The preliminary judgment result diagram of the sensitivity of ROX channel Sip gene detection in the present invention.

[0063] Fig.11 : When the concentration of the sample was 500 copies / mL, the sensitivity verification result of the present invention for Cfb gene detection in the Cy5 channel was shown.

[0064] Fig.12 : When the concentration of the sample was 500 copies / mL, the sensitivity verification result of the Sip gene detection of the ROX channel according to the present invention was shown.

[0065] Fig.13 : When the concentration of the sample was 1000 copies / mL, the sensitivity verification result of the present invention for Cfb gene detection in the Cy5 channel was shown.

[0066] Fig.14 : 1000 copies / mL, the present invention is for ROX channel Sip gene detection sensitivity verification result diagram.

[0067] Fig.15 : When the concentration is 2000 copies / mL, the sensitivity verification result of the present invention for Cfb gene detection in the Cy5 channel is shown.

[0068] Fig.16 : At 2000 copies / mL, the present invention shows the results of sensitivity verification of Sip gene detection for ROX channel. DETAILED DESCRIPTION

[0069] In the method for detecting group B streptococcus, usually a conserved sequence in group B streptococcus is selected to design primers and probes, and then the housekeeping gene in human genome DNA is combined to detect whether group B streptococcus in clinical samples. However, group B streptococcus suffers different evolutionary pressures in different human body environments and has different mutation rates. In order to reduce the missed detection of group B streptococcus caused by gene sequence mutation as much as possible, the present invention selects two sections of conserved sequences in group B streptococcus genome to design two groups of primers and probes, and simultaneously designs a group of primers and probes as internal control according to a section of conserved sequence in the streptococcus genome (such as this section of conserved sequence comes from 16S rRNA gene), and introduces human genome housekeeping gene (such as β-globin) as internal reference, establishes double quality control, double target verification system, so as to effectively avoid the abnormal results caused by a series of factors such as clinical sample sampling error, nucleic acid extraction error, and more importantly, this can also solve the problem of missed detection of group B streptococcus. Based on this, the designed primers and probes are as shown in Table 1.

[0070] Table 1. Primers and probes

[0071]

[0072] Table 1 contains a total of 4 TaqMan probes, each of which has a 5' end (x1, x2, x3 and x4) as a fluorescent reporter group, and a 3' end (y1, y2, y3 and y4) as a quencher group that emits or does not emit fluorescence. The fluorescent reporter groups of these 4 probes can be selected from FAM, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, ROX, Texas Red, LC RED640 and LCRED705, etc.; the quencher group can be selected from BHQ0, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse and NFQ, etc. The principle of selecting the quencher group is that the fluorescence absorption spectrum of the quencher group overlaps with the emission spectrum of the fluorescent reporter group. The preferred combination of the fluorescent reporter group and the quencher group of the four probes in Table 1 is any one of FAM-TARMA / BHQ1, VIC / HEX / JOE-TAMRA / BHQ1, ROX / Texas Red-BHQ2 and Cy5 / LC Red640-BHQ2 / BHQ3. Preferably, the fluorescent reporter group of each probe is different, so that four-channel detection can be performed simultaneously in a single tube.

[0073] The preferred fluorescent reporter groups are FAM, ROX and Cy5, because these three fluorescent reporter groups have the advantages of low background fluorescence and high fluorescence detection signal in fluorescent quantitative PCR detection.

[0074] Before performing fluorescent PCR, it is necessary to extract nucleic acid (DNA) from clinical samples. There are many methods for extracting nucleic acid, such as alkaline lysis, magnetic bead method and column extraction. For ease of explanation, the present invention uses alkaline lysis to extract nucleic acid from clinical samples, and the nucleic acid extracting solution used includes nucleic acid extracting solution 1 and nucleic acid extracting solution 2, wherein nucleic acid extracting solution 1 includes 10-100mM Tris, 10-100mM NaCl and 0.1%-2% SDS; nucleic acid extracting solution 2 includes 10-200mM NaCl, 10-100mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100. However, it should be noted that although alkaline lysis is used here to extract nucleic acid from clinical samples, the present invention is not limited to this nucleic acid extraction method, and methods such as magnetic bead method and column extraction can be used for the clinical sample nucleic acid extraction of the present invention.

[0075] For ease of explanation, in the following examples, the fluorescent reporter group and quencher group of the probe for detecting the 16S rRNA gene of Streptococcus are FAM-BHQ1, the fluorescent reporter group and quencher group of the probe for detecting the Sip gene of Group B Streptococcus are ROX-BHQ2, the fluorescent reporter group and quencher group of the probe for detecting the Cfb gene of Group B Streptococcus are CY5-BHQ3, and the fluorescent reporter group and quencher group of the probe for the human genome housekeeping gene β-globin are VIC-BHQ1. The strains used in the embodiments of the present invention are purchased from ATCC and are used to prepare the positive quality control and reference products in the kit.

[0076] The following examples further illustrate the present invention. These examples are not intended to limit the scope of the present invention, but to provide a further understanding of the present invention.

[0077] Example 1: Extraction of nucleic acid from clinical samples

[0078] For the convenience of explanation, the vaginal secretions of pregnant women are used as clinical samples for explanation in this embodiment.

[0079] Take 1 mL of vaginal secretion sample from pregnant women in a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, discard the supernatant, add 500 μL of nucleic acid extraction solution 1, shake and mix, centrifuge at 1200 rpm for 10 min, discard the supernatant; add 50 μL of nucleic acid extraction solution 2, shake and mix, place in a 100°C water bath or dry bath for 10 min, and use the supernatant as a DNA extraction solution for later use. Nucleic acid extraction solution 1 contains 50 mM Tris, 20 mM NaCl and 1% SDS; nucleic acid extraction solution 2 contains 50 mM Tris, 20 mM NaCl, 0.5% mercaptoethanol and 2% Chelex-100.

[0080] The nucleic acid extraction method in the present invention is alkaline lysis method, and a two-step method is used to wash and extract nucleic acid from clinical samples. The nucleic acid (DNA) extraction kit (magnetic bead method) (Zhehang Machinery No. 20150133) produced by Aikang Biotechnology (Hangzhou) Co., Ltd. is used as a control to extract nucleic acid from the same clinical samples, and the kit of the present invention is used to detect the human genome DNA housekeeping gene β-globin, and the detected Ct values ​​are compared. The results are as follows: Figure 1 and Figure 2 As shown. Figure 1 and Figure 2 It can be seen that the nucleic acid extraction method of the present invention has basically the same detection results as the magnetic bead extraction method, and at the same time has the characteristics of simple operation and low reagent cost.

[0081] Example 2: Detection of Group B Streptococcus using fluorescent PCR method

[0082] In this embodiment, a kit is used to perform fluorescent quantitative PCR detection on group B streptococci, and the kit includes a PCR reaction solution, an enzyme mixture, a positive quality control, and a negative quality control. Preferably, this kit may also include nucleic acid extract 1 and nucleic acid extract 2 in Example 1. The PCR reaction solution preparation system is shown in Table 2. The enzyme mixture preparation system is shown in Table 3. The positive quality control is a group B streptococcus culture, and the negative quality control is a 0.9% NaCl solution.

[0083] Table 2. PCR reaction solution preparation system table (1 person)

[0084]

[0085] Table 3. Enzyme mixture preparation system (for one person)

[0086]

[0087] When detecting pathogen group B streptococci in clinical samples, the PCR reaction solution and the enzyme mixture need to be mixed, and the required number of samples n [n = number of clinical samples + 1 tube of positive control + 1 tube of negative control] is calculated. 36 μL of PCR reaction solution is added to different reaction tubes, and then 4 μL of the DNA solution extracted in Example 1, 4 μL of negative control and 4 μL of positive control are added to different reaction tubes respectively.

[0088] Place the reaction tubes in a certain order on a fluorescent PCR instrument (ABI 7500) and perform PCR amplification according to the following procedure. The amplification procedure is shown in Table 4:

[0089] Table 4. Fluorescence PCR amplification program

[0090]

[0091] After PCR amplification, the presence of group B streptococci was detected based on the Ct values ​​of the FAM channel, VIC channel, ROX channel, and Cy5 channel. The detection results are shown in Table 5.

[0092] Table 5. Cutoff value of test results and result interpretation

[0093]

[0094] Common test results of clinical samples are shown in Table 6. Other abnormal results need to be interpreted in conjunction with clinical findings.

[0095] Table 6. Common interpretation of results for clinical samples

[0096]

[0097]

[0098] Example 3: Internal clinical sample validation

[0099] Clinical samples were collected from Hangzhou Aidikang Medical Testing Center, a total of 300 pregnant and postpartum women's vaginal secretion samples for internal clinical evaluation.

[0100] Nucleic acids were extracted from 300 samples of cervical secretions of pregnant women according to the method in Example 1, and then the nucleic acids in these 300 samples were amplified by fluorescent PCR according to the method in Example 2. The results were recorded as the results of Experimental Group 1.

[0101] According to the method described in Example 2, the Cfb gene (experimental group 2) was detected using the sequence SEQ ID NO: 7-9, and the Sip gene (experimental group 3) was detected using the sequence SEQ ID NO: 4-6, respectively, and the internal standard gene detection was combined to prepare the PCR reaction solution preparation system for group B streptococcus, and the nucleic acid in the same 300 pregnant women's cervical secretion samples was amplified by fluorescent PCR in parallel, and the test results were recorded. The analysis of the three groups of test results is shown in Table 7.

[0102] Table 7: Consistency analysis of dual-target system and single-target detection results

[0103]

[0104] In the above results, the test results of experimental group 2 and experimental group 3 each had one sample whose result was not the same as that of experimental group 1. The samples with inconsistent results were verified by Sanger sequencing, and the verification results were consistent with the test results of experimental group 1, which showed that the dual-target detection system adopted by the present invention can effectively avoid the missed detection of positive samples, thereby improving the detection accuracy.

[0105] To further verify the accuracy of the dual-target detection system, the group B streptococcal nucleic acid detection reagent (fluorescence PCR method) (primer probe designed for Cfb gene, upstream primer: 5'-AGCAATCACACATGCTGTTGGA-3', downstream primer: 5'-TAATGCTGTTTGAAGTGCTGCT-3', probe: 5'FAM-CAGTTGAATCCAAATGTTACGGTACAAC-TAMRA 3', hereinafter referred to as control reagent 1) and group B streptococcal nucleic acid determination reagent (fluorescence PCR method) (primer probe designed for Sip gene, hereinafter referred to as control reagent 2, upstream primer: 5'-TTGACATCGACAATGGCAGC-3', downstream primer: 5'-TAACACTTGCCACTCTAGGG-3', probe: 5'FAM-AACAGATACGACGTGGACAG-TAMRA 3', hereinafter referred to as control reagent 2) was also used as a control reagent to test the nucleic acid in the same 300 pregnant women's cervical secretion samples in parallel. The test results are shown in Table 8.

[0106] Table 8. Clinical sample test results

[0107]

[0108] The results in Table 8 show that the results of 2 samples in control reagent 1 are not the same as those in the present invention; the results of 1 sample in control reagent 2 are not the same as those in the present invention. The samples with inconsistent results were verified by Sanger sequencing, and the verification results were consistent with the detection results of experimental group 1, which shows that the dual-target detection system adopted by the present invention can effectively avoid the missed detection of positive samples, thereby improving the detection accuracy.

[0109] Example 4 Establishment and evaluation of reference product system

[0110] In this example, the following reference samples were set for the kit of the present invention to evaluate its performance. Positive reference sample setting: Group B Streptococcus (serotypes Ⅰa, Ⅰb, Ⅱ, Ⅲ and Ⅴ) with a concentration of about 5×10 5 The standard strain culture with 100 copies / mL was used as the positive reference of the kit (P1-P5). Negative reference setting: 1 sample of Chlamydia trachomatis, 1 sample of Neisseria gonorrhoeae, 1 sample of Ureaplasma urealyticum, 1 sample of HSV-2, 4 samples of HPV (including 1 each of HPV16, 18, 52, and 58), 1 human genomic DNA, and 1 TE buffer were selected as negative references (recorded as N1-N10 in sequence).

[0111] The positive and negative coincidence rate of the reference product provided in the kit was evaluated according to the method described in Example 2. The results are shown below:

[0112] The test results of positive reference materials P1 to P5 were positive in FAM, ROX, and Cy5 channels, and negative in VIC channel. Figures 3 to 7 shown.

[0113] Negative references N1 to N9, a total of 9 negative references, all showed negative results in FAM, ROX, and Cy5 channels, and only showed positive results in VIC channel. The negative reference N10 showed negative results in FAM, VIC, ROX, and Cy5 channels. Figure 8 shown.

[0114] Example 5 Detection Sensitivity Analysis

[0115] In this example, the most common serotype III of group B streptococcus was cultured with a high concentration of standard strains (about 10 5 The samples were diluted to half the amount of 1000 copies / mL, and the gradient dilution samples were tested according to the method in Example 2. The detection limit of the kit was preliminarily determined to be around 1000 copies / mL. Figures 9-10 shown.

[0116] According to the kit described in Example 2, the test was repeated 20 times on different concentrations of the culture products of the most common serotype III high-concentration standard strain of group B Streptococcus (the concentrations were 500, 1000, and 2000 copies / mL, respectively), and the detection limit of the kit was confirmed with a detection rate of 95%. The test results are detailed in Tables 8 and 9, wherein Table 8 is the statistical results of the ROX channel detection, and Table 9 is the statistical results of the Cy5 channel detection. The detection rates of the Sip gene amplification primer probe for 500, 1000, and 2000 copies / mL standard bacteria were 80%, 100%, and 100%, respectively, while the detection rates of the Cfb gene amplification primer probe for 500, 1000, and 2000 copies / mL standard bacteria were 80%, 95%, and 100%, respectively; in summary, the detection limit of the kit is 1000 copies / mL. Figures 11 to 16 shown.

[0117] Table 9. Statistics of the lower limit of detection of Sip gene in ROX channel

[0118]

[0119]

[0120] Table 10. Statistics of the lower limit of detection of Cfb gene in Cy5 channel

[0121]

[0122] Example 6 Detection Precision Analysis

[0123] According to the kit described in Example 2, one medium-concentration positive sample (greater than 10 5 The tests were repeated 8 times for 15 samples, 1 weak positive sample (about 2000 copies / mL), and 1 negative sample, and the test results of different channels were statistically analyzed, as shown in Table 10. From the results, it can be concluded that the CV of the Ct value detected by the kit is less than 5%, which shows that the kit of the present invention has good intra-batch precision.

[0124] Table 11. Precision sample test results

[0125]

[0126] Sequence Listing <110> Lido (Hong Kong) Co., Ltd. <120> Methods, oligonucleotides and kits for detecting Streptococcus <150> 201911146519.X <151> 2019-11-21 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <400> 1 tcttaaagcc agtctcagtt cggat 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 gacttcgggt gttacaaact ctcgt 25 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <400> 3 tgtaggctgc aactcgccta catgaagtc 29 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <400> 4 tgttgcagac caaaaagttt ctctc 25 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <400> 5 cttgtgctaa tacttctttt gatttcaaa 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <400> 6 ctggcgcaga agaatatgtc ttcattggc 29 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 aacttgttgt accgtaacat ttggat 26 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <400> 8 agtacttaac gtcaaagaat ttaaagt 27 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <400> 9 caactgaact ccaacagcat gtgtgattgc 30 <210> 10 <211> twenty four <212> DNA <213> Artificial Sequence <400> 10 agacaatggt gcatctgact cctg 24 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 ccaccaactt catccacgtt cac 23 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <400> 12 tggagaagtc tgccgttatgccctgt 27 <210> 13 <211> twenty two <212> DNA <213> Artificial Sequence <400> 13 agcaatcaca catgctgttg ga 22 <210> 14 <211> twenty two <212> DNA <213> Artificial Sequence <400> 14 taatgctgtt tgaagtgctg ct 22 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <400> 15 cagttgaatc caaatgttac ggtacaac 28 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 ttgacatcga caatggcagc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 taacacttgc cactctaggg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 aacagatacg acgtggacag 20

Claims

1. An oligonucleotide for detecting streptococci, characterized in that The oligonucleotides include (1) primers and probes for detecting Streptococcus, whose base sequences are SEQ ID NOs: 1 to 3 respectively; (2) primers and probes for detecting Sip gene of group B Streptococcus, whose base sequences are SEQ ID NOs: 4 to 6 respectively; (3) primers and probes for detecting Cfb gene of group B Streptococcus, whose base sequences are SEQ ID NOs: 7 to 9 respectively.

2. The oligonucleotide according to claim 1, characterized in that The oligonucleotide also includes (4) primers and probes for detecting internal reference genes, and their base sequences are SEQ ID NOs: 10 to 12, respectively.

3. The oligonucleotide according to claim 1, characterized in that The fluorescent reporter and quencher groups of each probe were selected from any one of FAM-TARMA / BHQ1, VIC / HEX / JOE-TAMRA / BHQ1, ROX / Texas Red-BHQ2, and Cy5 / LC Red640-BHQ2 / BHQ3.

4. The oligonucleotide according to claim 3, characterized in that Each probe has a different fluorescent reporter gene.

5. A kit for detecting streptococci, the kit comprising a fluorescent PCR reaction solution, characterized in that: The fluorescent PCR reaction solution comprises the oligonucleotide as shown in any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit also includes a positive quality control and a negative quality control, the positive quality control is a group B streptococcus culture, and the negative quality control is a 0.9% NaCl solution.

7. The kit as claimed in claim 5, further comprising a nucleic acid extracting solution 1 and a nucleic acid extracting solution 2, characterized in that: The nucleic acid extract 1 comprises 10-100 mM Tris, 10-100 mM NaCl and 0.1%-2% SDS, and the nucleic acid extract 2 comprises 10-200 mM NaCl, 10-100 mM Tris, 0.1-2% mercaptoethanol and 1-5% Chelex-100.

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