Loop-mediated isothermal amplification primer for detection of vibrio parahaemolyticus and use thereof

Abstract

Primers and primer sets for amplifying DNA of Vibrio parahaemolyticus and methods of detecting Vibrio parahaemolyticus using the primers and primer sets.

Description

[0001] sequence list

[0002] This application includes a sequence list submitted electronically to the U.S. Patent and Trademark Office via EFS-Web as an ASCII text file entitled “82721WO003_ST25.txt”, which is 1.26 kilobytes in size and was created on December 15, 2020. Information contained in the sequence list is incorporated herein by reference. Background Technology

[0003] Some foodborne illnesses, such as vibriosis, are caused by Vibrio parahaemolyticus. parahaemolyticus This can be caused by consuming foods containing microorganisms, such as shellfish.

[0004] Loop-mediated isothermal amplification (LAMP) is a method for amplifying DNA described in, for example, WO0028082, WO0134790, and WO0177317. The amplified DNA was detected using a Molecular Detection kit obtained from 3M (St. Paul, MN). Detailed Implementation

[0005] Throughout this disclosure, for convenience, the singular forms such as “a,” “an,” and “the” are often used; however, unless the context explicitly specifies or clearly indicates that it is singular only, the singular forms are intended to include the plural. When referred to solely in the singular, the term “only one” is commonly used.

[0006] Some terms used in this disclosure are defined as follows. Other terms will be familiar to those skilled in the art and should be given the meanings that those skilled in the art would assign to them.

[0007] The terms “common,” “typical,” and “usual,” as well as “commonly,” “typically,” and “usually” are used herein to refer to features frequently employed in this invention and are not intended to indicate, unless explicitly used in contrast to the prior art, that these features are present in the prior art in far fewer ways than those common, usual, or typical features in the prior art.

[0008] The term "LAMP" is an acronym for loop-mediated isothermal amplification, a method for amplifying DNA that has been described in, for example, WO0028082, WO0134790, and WO0177317.

[0009] Microbial detection is achieved by amplifying DNA fragments specific to the type of microorganism to be detected, and then detecting the amplified DNA. If the amplified DNA is present at detectable levels, it indicates the presence of the microorganism of interest. If the DNA is absent, it indicates the absence of the DNA of interest, and therefore the absence of the microorganism of interest. PCR is a well-known method that relies on thermal cycling to amplify DNA. LAMP is another method used to amplify DNA. Unlike PCR, LAMP occurs at a constant high temperature (typically around 60°C, such as 50°C to 70°C), thus eliminating the need for thermal cycling.

[0010] In short, LAMP requires four different primers, but six primers are often used. The required primers are two LAMP primers and two substitution primers. Optional primers are two loop primers. LAMP primers have a 3' fragment that binds to a specific target sequence and a 5' fragment that is inversely complementary to the inner target sequence. Extension from the substitution primers produces a primary amplicon and forms a self-absorbing loop structure. Optional loop primers bind within the loop structure to facilitate exponential amplification. The primer set used for amplification consists of two LAMP primers (called LampF and LampB), two substitution primers (called DisF and DisB), and optionally one or two loop primers (called LoopF ​​and LoopB).

[0011] In each primer set, at least two LAMP primers are not found in nature. This is because one end of each LAMP primer is complementary to a DNA fragment on the forward strand of the template, and the other end of each LAMP primer is complementary to a discontinuous DNA fragment on the reverse strand of the template. Furthermore, at least the 5' primer or primer fragment of each LAMP primer is the reverse complementary sequence of the target DNA, which is also not found in nature.

[0012] After amplification, the DNA can be detected using various methods, including real-time bioluminescence (BART). The 3M Molecular Detection System (purchased from 3M Company, St. Paul, MN, USA) is a commercially available system that uses BART to detect microorganisms after LAMP amplification of the microbial DNA.

[0013] The problem is that no known primers exist that can be used for LAMP amplification of a unique and specific portion of the Vibrio parahaemolyticus nuclear DNA for identification of Vibrio parahaemolyticus microorganisms. Specifically, the problem can be formulated as how to identify any strain of any of the various strains of Vibrio parahaemolyticus without obtaining false positives by mistakenly identifying another species of Vibrio as a Vibrio parahaemolyticus. Another problem is that Vibrio parahaemolyticus cannot currently be detected by LAMP amplification followed by a detection method. More specifically, primers for rapid amplification and detection, such as those for time intervals of no more than 48 hours, no more than 24 hours, no more than 12 hours, no more than 6 hours, no more than 4 hours, no more than 2 hours, no more than 90 minutes, or even no more than 60 minutes, are unavailable.

[0014] Another challenge is identifying a suitable gene for the specific detection of Vibrio parahaemolyticus without needing to detect other Vibrio species. Another challenge is finding a use for the VP175 gene, particularly for the detection of Vibrio parahaemolyticus. The gene's potential target for Vibrio parahaemolyticus detection is currently unknown. A related challenge is finding a LAMP primer set that targets the VP175 gene for the detection of Vibrio parahaemolyticus.

[0015] In summary, as a solution to one or more of these problems and others, this disclosure provides primers that can be used to amplify Vibrio parahaemolyticus via the LAMP method. The primers can specifically detect the VP175 gene of Vibrio parahaemolyticus.

[0016] A primer set for Vibrio parahaemolyticus may include Lamp primers, particularly the Vibrio parahaemolyticus LampF primer having SEQ ID NO:1. Optionally, the Vibrio parahaemolyticus LampF primer may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:1.

[0017] Any suitable replacement primer for Vibrio parahaemolyticus can be used in conjunction with the LampF primers described above in the primer set. A suitable replacement primer is the Vibrio parahaemolyticus DisF primer having the sequence of SEQ ID NO:2. Optionally, the Vibrio parahaemolyticus DisF primer may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:2. In principle, other replacement primers can be used in conjunction with Lamp primers, such as the Lamp primers discussed above.

[0018] The primer set disclosed in this invention for use with Vibrio parahaemolyticus may optionally include suitable loop primers. A suitable loop primer is the Vibrio parahaemolyticus LoopF ​​primer having the sequence of SEQ ID NO:2. Optionally, the Vibrio parahaemolyticus LoopF ​​primer may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:3. In principle, other loop primers may be used.

[0019] The primer set for Vibrio parahaemolyticus may include (usually in addition to the Vibrio parahaemolyticus LampF primers described above, but in some cases as an alternative) Vibrio parahaemolyticus LampB primers having the sequence of SEQ ID NO:4. Optionally, the Vibrio parahaemolyticus LampB primers may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:4.

[0020] Any suitable replacement primer for Vibrio parahaemolyticus can be used in a primer set together with one or both of the aforementioned lamp primers, i.e., Vibrio parahaemolyticus LampF primer, Vibrio parahaemolyticus LampB primer, or both Vibrio parahaemolyticus LampF primer and Vibrio parahaemolyticus LampB primer. A suitable replacement primer is the Vibrio parahaemolyticus DisB primer having the sequence of SEQ ID NO:5. Optionally, the Vibrio parahaemolyticus DisB primer may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:5. Other replacement primers may be used in principle.

[0021] The primer set for Vibrio parahaemolyticus may optionally include suitable circular primers. A suitable circular primer is the stx1 all var LoopB primer having the sequence of SEQ ID NO:6. Optionally, the stx1 all var LoopB primer may have a sequence having at least 99%, at least 95%, at least 90%, at least 85%, or at least 80% homology with SEQ ID NO:6. Other circular primers may be used in principle.

[0022] A summary of the primers and primer sets disclosed in this paper can be found in Table 1.

[0023] Table 1

[0024]

[0025] Any primer set described herein may be in the form of lyophilized granules or powder, which may optionally contain other substances. Examples of other substances that may be included in lyophilized granules or powder include sugars (such as glucose), lyophilization aids, preservatives, antioxidants, etc.

[0026] In practice, any of the aforementioned primer sets can be used in methods for amplifying target DNA. In such methods, the target DNA is exposed to any of the aforementioned primer sets under conditions suitable for amplifying the target DNA. The target DNA is typically DNA from Vibrio parahaemolyticus organisms, and particularly DNA from the VP175 gene of Vibrio parahaemolyticus. While suitable conditions for amplifying target DNA with primers are known or readily determined by a technician, specific conditions that can be used include temperatures ranging from 50°C to 70°C.

[0027] The method may further include detecting target DNA. Methods for detecting target DNA are known. One exemplary method requires the use of a 3M molecular detection system. Other methods may also be used.

[0028] Example

[0029] Bioinformatics and primer design

[0030] Complete genomes available from NCBI (National Center for Biotechnology Information) were compiled, and whole-genome alignment was performed one chromosome / replicon at a time using the advanced Mauve algorithm of the Mauve Genome Aligner. Based on the similarity map generated by Mauve, manual scanning and alignment were performed for orthologous genes with moderate to high levels of conservation.

[0031] The BLAST (Basic Local Alignment Search Tool) algorithm was used to search for selected genes against the NCBI nucleotide collection (nr / nt) for comparison of primary biological sequence information. Nucleic acid identity percentage was determined as the worst inclusion match, and the best inclusion match was defined as having over 100% coverage. Multiple sequence alignments were generated in BioEdit using Clustal W for the highest-scoring gene candidates. All available Vibrio parahaemolyticus sequences from the NCBI nr / nt and refseq genome databases were included in the alignments.

[0032] Regions suitable for LAMP primer design were identified, and specificity was further validated by BLAST analysis against the NCBI non-redundant (nr / nt) nucleotide database. Table 2 shows a set of LAMP primers designed for the detection of the VP175 gene.

[0033] Table 2

[0034]

[0035] Cultivation methods

[0036] Cultures identified in Table 3 were obtained from the BEI Resource Bank (BEI) or the American Type Culture Collection (ATCC) and propagated by streaking frozen cultures onto trypsin-soybean agar plates and incubating at 32°C for 24 hours. Colonies were suspended in 10 mL of buffered peptone water broth (BPW-ISO, available from 3M Company, St. Paul, MN, US) using a 10 µL inoculation loop. Cultures were grown at 32°C for 18 hours and were noticeably turbid after incubation.

[0037] After incubation, dilute the culture to 1×10^6 CFU / mL in BPW-ISO. Allocate 20 µl of each dilution to lysis buffer (available from 3M, as per 3M labelling instructions). ™ Part of Molecular Detection Assay 2 – Cronobacter sakazakii Kit). Heat the sample to 100°C for 15 minutes, then cool for 5 minutes. Then, dispense 20 µl of each sample into a 3M solution. ™ Molecular Detection Assay 2 – Cronobacter sakazakii Kit in test tubes.

[0038] Add 2 µl of a 10x primer mixture containing 4 mM MgSO4 (New England Biolobs, Ipswich, MA, US) to a test tube and mix up and down five times using a multichannel pipette. In each tube, the final concentration of each LAMP primer was 0.8 µl; the final concentration of each LOOP primer was 0.4 µl; the final concentration of each DIS primer was 0.2 µl; and the final concentration of MgSO4 was 0.4 mM.

[0039] Use 3M ™ The Molecular Detection System 2 instrument performed a 60-minute test at 60°C to determine the presence of each sample. The results are shown in Table 3, where a positive result indicates the presence of an organism, and a negative result indicates the absence of an organism.

[0040] Table 3

[0041] sequence list <110> 3M Innovative Properties Company <120> Loop-mediated isothermal amplification primers for Vibrio parahaemolyticus detection and their applications <130> 82721WO003 <150> 62 / 977,586 <151> 2020-02-17 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 43 <212> DNA <213> artificial <220> <223> Synthetic <400> 1 tcgctattac cttggcaacg cttctcaaca aagacatgaa tga 43 <210> 2 <211> 42 <212> DNA <213> artificial <220> <223> Synthetic <400> 2 gcttctaaga tgtggaacat ctgcttggat aattggctat gc 42 <210> 3 <211> 18 <212> DNA <213> Vibrio parahaemolyticus <400> 3 gatgtccgtc aacagcac 18 <210> 4 <211> twenty two <212> DNA <213> Vibrio parahaemolyticus <400> 4 actcaaagct tatctctttg gt 22 <210> 5 <211> 19 <212> DNA <213> Vibrio parahaemolyticus <400> 5 ccatggctca agagttcat 19 <210> 6 <211> 19 <212> DNA <213> Vibrio parahaemolyticus <400> 6 ggcgactgaa cgatttagg 19

Claims

1. A primer set comprising primers as shown in SEQ ID NO: 1 and primers as shown in SEQ ID NO:

4.

2. The primer set according to claim 1, wherein the primer set further comprises primers as shown in SEQ ID NO:

2.

3. The primer set according to claim 1, wherein the primer set further comprises primers as shown in SEQ ID NO:

3.

4. The primer set according to claim 1, wherein the primer set further comprises primers as shown in SEQ ID NO:

5.

5. The primer set according to claim 1, wherein the primer set further comprises primers as shown in SEQ ID NO:

6.

6. A lyophilized powder comprising a primer set according to any one of the preceding claims.

7. A granule comprising the freeze-dried powder according to claim 6.

8. A method for amplifying target DNA in food, the method comprising exposing the target DNA to a primer set according to any one of claims 1-5, a lyophilized powder according to claim 6, or granules according to claim 7 under conditions sufficient to form amplified target DNA.

9. The method of claim 8, wherein the conditions include a temperature of 50°C to 70°C.

10. The method of claim 8, further comprising detecting the presence of amplified target DNA.

11. The method according to claim 8, wherein the target DNA is Vibrio parahaemolyticus (V. parahaemolyticus). V. parahaemolyticus DNA.

12. The method of claim 8, wherein the target DNA is the DNA of Vibrio parahaemolyticus VP175.

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