Primer and probe combination, kit and method for detecting gastrointestinal pathogenic bacteria

Abstract

The invention relates to a primer and probe combination, a kit and a method for detecting gastrointestinal tract pathogenic bacteria. The gastrointestinal pathogenic bacteria comprise vibrio cholerae, vibrio parahaemolyticus, salmonella, shigella, diarrheagenic escherichia coli, campylobacter, yersinia enterocolons, clostridium difficile, cronobacter sakazakii, aeromonas hydrophila, plesiomonas shigelloides and vibrio fluvialis. The primer and the probe group can be independently used for PCR amplification detection and can be combined in the same reaction system and reaction procedure for multiple PCR amplification detection, and the primers and the probes aiming at different pathogenic bacteria cannot interfere with each other during multiple PCR amplification detection. The primer and probe combination covers various types of gastrointestinal pathogenic bacteria, and is high in sensitivity, low in detection limit, good in specificity and wide in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and in particular relates to a primer and probe combination, a kit and a method for detecting gastrointestinal pathogenic bacteria. Background Art

[0002] The most common clinical symptom after infection with gastrointestinal pathogens is diarrhea, which can be caused by a variety of reasons, including non-infectious factors such as chemicals, drugs, enteritis, allergies and radiotherapy, and infectious factors such as viruses, bacteria, fungi and parasites. Among the diarrhea caused by gastrointestinal pathogens, common pathogens include Vibrio cholerae (VC), Vibrio Parahaemolyticus (VP), Salmonella enteriditis (SE), Shigella Castellani (SC), diarrhea Escherichia coli (DEC), Campylobacter (CA), Yersinia enterocolitica (YE), Clostridioides difficile (CD), Enterobacter sakazakii (ES), Aeromonas hydrophila (AH), Plesiomonas Shigelloides (PS), Vibrio fluvialis (VF), etc. These bacteria can be transmitted through contaminated water or food, causing acute gastrointestinal inflammation with symptoms such as nausea, vomiting, abdominal pain and diarrhea, and in severe cases may lead to dehydration and electrolyte imbalance.

[0003] Diarrhea caused by gastrointestinal pathogens is a global public health problem with a high incidence rate. However, infectious diarrhea is also a preventable and treatable disease. According to the latest data from the World Health Organization (WHO), diarrheal disease is the third leading cause of death in children aged 1-59 months. Approximately 443,832 children under the age of 5 die from diarrhea each year, and another 50,851 children aged 5 to 9 die from the disease. There are nearly 1.7 billion cases of childhood diarrhea worldwide each year, indicating that the global incidence of diarrhea is very high. With the development of society and prevention technology, the global diarrhea mortality rate per 100,000 children under the age of 5 decreased by 70.01% between 1990 and 2023, with the largest contribution coming from reduced exposure to unsafe sanitation facilities (reduced by 14.12%) and improvements in child wasting (reduced by 9.82%) and the use of oral rehydration salts (increased by 7.01%). In previous studies, it was found that infectious diarrhea showed a specific spatiotemporal distribution, usually clustered in different geographical locations in space. Therefore, monitoring diarrheal diseases and identifying pathogens remain a top priority.

[0004] Research related to laboratory diagnosis of diarrheal diseases caused by gastrointestinal pathogens has focused on improving the accuracy, efficiency and coverage of testing. Diagnostic tests include blood tests, stool tests, abdominal CT and PCR. Blood tests check the levels of electrolytes and serum creatinine. Routine tests may indicate severe diarrhea with fever or toxicity in patients. Leukocytosis or neutrophil nuclear shift to the left indicates inflammation caused by bacterial pathogens and has prognostic significance in Clostridium difficile-associated diarrhea. Stool tests are expensive to determine the cause of diarrhea and are not necessary in most cases of non-severe diarrhea. Stool samples for patients at high risk for diarrhea or to determine which pathogen is infected should be retained after evaluation to determine the cause. Abdominal CT flexible sigmoidoscopy or colonoscopy has limited value in the routine evaluation of patients with acute diarrhea. Flexible sigmoidoscopy is a useful diagnostic method in cases of persistent diarrhea and selected cases of acute diarrhea with unclear clinical colitis, such as suspected Clostridium difficile-associated diarrhea with negative stool toxins. PCR diagnostic testing is currently the most widely used laboratory technology, often combined with a single test to detect multiple enteric pathogens. PCR has the advantage of high sensitivity, but it focuses on genes rather than virulence factors.

[0005] PCR detection methods include ordinary PCR, constant temperature PCR technology and real-time fluorescence quantitative PCR technology. Ordinary PCR technology cannot be quantified and has low sensitivity and accuracy. Since the lid needs to be opened during the detection process, there is a risk of cross-contamination. Constant temperature PCR technology has problems with multiplicity and specificity, and its detection ability for complex samples is limited. Real-time fluorescence quantitative PCR technology can directly detect changes in fluorescent signals during PCR amplification, and achieve high sensitivity and specificity detection of specific nucleic acid sequences. The entire amplification process is a closed-tube operation, and the entire process from sample addition to result acquisition is highly automated, reducing the complexity of manual operation and possible errors. Real-time fluorescence quantitative PCR technology is widely used in the fields of DNA and RNA quantification, gene expression research, pathogen detection, genetic disease analysis, tumor research, etc. By monitoring and mapping the pathogen spectrum of infectious diarrhea, major pathogens such as norovirus are discovered, and data support is provided for public health decision-making.

[0006] There are certain defects in the patent technologies related to gastrointestinal pathogens that have been disclosed so far. For example, the Chinese patent application CN116334264A adopts an electrochemical method. This method is greatly affected by temperature. Temperature changes during the test process will have a great impact on the performance of the reagent. The electrochemical chip is difficult to process and has a high cost for one-time use. In addition, the electrochemical sensor can only test high-concentration samples and has low sensitivity to low-concentration samples. Chinese patent applications CN103074450A, CN107164562A and CN107245531A are based on capillary electrophoresis. This method has high technical requirements and requires professional technicians to operate capillary electrophoresis equipment. In addition, the equipment cost is high and the injection deviation is large. Although a variety of pathogenic diarrhea pathogens have been detected, the reagent sensitivity is low and it is easy to miss detection, which is not conducive to the use of the public health system. Chinese patent applications CN107164562A and CN117025852A only detect common diarrhea viruses in the patents, and do not identify diarrhea pathogens. Chinese patent application CN106916906A adopts loop-mediated isothermal amplification technology, which has high false positives and uses 8 amplification reactions to detect 6 pathogen systems, with high reagent costs. Chinese patent CN103911443A applies for the use of gene chip method, which requires amplification before reading the chip, and requires the purchase of amplifiers and chip scanners. The instrument cost is high and the operation is complicated. The entire detection process requires the preparation of 3 reaction systems, which is not conducive to market promotion and use. In the application of Chinese patent CN104662167A, primers and probes related to the detection of diarrhea-related pathogens are disclosed, including enterohemorrhagic Escherichia coli EHEC, enteroinvasive Escherichia coli EIEC, enteroaggregative Escherichia coli EPEC, enterotoxigenic Escherichia coli ETEC, enteroaggregative Escherichia coli EAEC), Salmonella, Campylobacter jejuni, Campylobacter coli, Yersinia, Vibrio cholerae, Giardia lamblia, Entamoeba histolytica and Cryptosporidium. The detection of diarrheal Escherichia coli is divided into 5 types for separate detection.

[0007] Blood tests, stool tests, abdominal CT and other methods on the market for gastrointestinal pathogenic bacteria diseases have certain problems in terms of timeliness, complexity of operation, sensitivity and specificity. The common reagent fluorescent PCR method on the market has shortcomings such as incomplete targets or other targets are extremely rare in domestic clinical practice. It cannot meet the domestic regulatory agencies' monitoring of gastrointestinal pathogens. At the same time, the fluorescent PCR reagents on the market have low sensitivity and poor specificity. Summary of the invention

[0008] Based on this, the purpose of the present invention is to provide a primer and probe combination for detecting gastrointestinal pathogens and its use in preparing a kit. The primer and probe group provided by the present invention can realize simultaneous detection of 12 common gastrointestinal pathogens on the same detection platform. The detection range covers multiple types of gastrointestinal pathogens, and has high sensitivity, low detection limit and good specificity.

[0009] The first aspect of the present invention is to provide a primer and probe combination for detecting gastrointestinal pathogens, wherein the primer and probe combination comprises at least two of the following primers and probes for 12 gastrointestinal pathogens:

[0010] Primers and probes for Vibrio cholerae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.1, the reverse primer shown in SEQ ID NO.2, and the probe shown in SEQ ID NO.3;

[0011] Primers and probes for Vibrio parahaemolyticus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.10, the reverse primer shown in SEQ ID NO.11, and the probe shown in SEQ ID NO.12;

[0012] Primers and probes for Salmonella, whose nucleotide sequences are the forward primer shown in SEQ ID NO.25, the reverse primer shown in SEQ ID NO.26, and the probe shown in SEQ ID NO.27;

[0013] Primers and probes for Shigella, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.31, the reverse primer shown in SEQ ID NO.32, and the probe shown in SEQ ID NO.33;

[0014] Primers and probes for diarrheagenic Escherichia coli, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.37, the reverse primer shown in SEQ ID NO.38, and the probe shown in SEQ ID NO.39;

[0015] Primers and probes for Campylobacter, whose nucleotide sequences are the forward primer shown in SEQ ID NO.49, the reverse primer shown in SEQ ID NO.50 and the probe shown in SEQ ID NO.51;

[0016] Primers and probes for Yersinia enterocolitica, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.55, the reverse primer shown in SEQ ID NO.56, and the probe shown in SEQ ID NO.57;

[0017] Primers and probes for Clostridium difficile, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.70, the reverse primer shown in SEQ ID NO.71, and the probe shown in SEQ ID NO.72;

[0018] Primers and probes for Cronobacter sakazakii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.76, the reverse primer shown in SEQ ID NO.77, and the probe shown in SEQ ID NO.78;

[0019] Primers and probes for Aeromonas hydrophila, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.82, the reverse primer shown in SEQ ID NO.83, and the probe shown in SEQ ID NO.84;

[0020] Primers and probes for Pleistomonas shigellae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.94, the reverse primer shown in SEQ ID NO.95 and the probe shown in SEQ ID NO.96;

[0021] The primers and probes for Vibrio fluvibrio have nucleotide sequences as shown in SEQ ID NO.106 for the forward primer, SEQ ID NO.107 for the reverse primer and SEQ ID NO.108 for the probe.

[0022] In some embodiments, the primer and probe combination includes: primers and probes for the Salmonella, and primers and probes selected from at least one of Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like bacteria, and Vibrio fluvial.

[0023] In some embodiments, the primer and probe combination includes primers and probes for the Salmonella and Shigella and diarrheagenic Escherichia coli; or,

[0024] The primers and probes for the Salmonella and Campylobacter and Yersinia enterocolitica; or,

[0025] The primers and probes for the Salmonella, Clostridium difficile and Cronobacter sakazakii; or,

[0026] The primers and probes for the Salmonella and for Aeromonas hydrophila and Shigella oxytoca.

[0027] In some embodiments, the primer and probe combination includes primers and probes for the Salmonella and for Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like bacteria and Vibrio fluvii.

[0028] In some embodiments, the primer and probe combination also includes primers and probes for an internal standard gene; preferably, the internal standard gene is an MS2 phage gene fragment, and the primers and probes for the internal standard gene are nucleotide sequences such as a forward primer shown in SEQ ID NO.109, a reverse primer shown in SEQ ID NO.110, and a probe shown in SEQ ID NO.111.

[0029] In some embodiments, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; the fluorescent group is selected from FAM, VIC, CY5 or ROX, and the quenching group is selected from BHQ2, BHQ3, MGB or BHQ1.

[0030] The second aspect of the present invention is to provide a kit for detecting gastrointestinal pathogens, the kit comprising the primer and probe combination as described above.

[0031] The third aspect of the present invention is to provide a method for detecting gastrointestinal pathogens, comprising the following steps: taking DNA from a sample to be tested, and performing multiplex PCR amplification using the primer and probe combination described above, or the kit described above.

[0032] In some of the embodiments, the reaction system of the multiplex PCR amplification includes the following components: Tris-HCl, Tween-20, BSA, MgCl2, dNTPs, DNA polymerase, UNG enzyme, the primer and probe combination.

[0033] In some embodiments, the concentration of the primers for Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Pseudomonas shigellae and Vibrio fluvii in the reaction system is 150nM to 500nM, preferably 150nM to 300nM, and more preferably 150nM to 200nM; and / or,

[0034] The concentrations of the probes for Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like bacteria and Vibrio fluvii in the reaction system are each 125nM to 300nM, preferably 125nM to 200nM, and more preferably 125nM to 150nM.

[0035] In some of the embodiments, the reaction procedure of the multiplex PCR amplification is: 1) 95° C., 60 s; 2) 95° C., 5 s; 60° C.; 25 s; 40 cycles.

[0036] After extensive research and screening, the present invention has obtained a set of primers and probes for detecting 12 common gastrointestinal pathogens. The primers and probes can be used alone for PCR amplification detection, and can be combined in the same reaction system and reaction program for multiple PCR amplification detection. During multiple PCR amplification detection, the primers and probes for different pathogens will not interfere with each other. The primer and probe combination of the present invention covers multiple types of gastrointestinal pathogens, and has high sensitivity, low detection limit and good specificity.

[0037] The detection method of the present invention is simple to operate and low in cost. It solves the current problems of incomplete detection of gastrointestinal pathogens, high cost and low sensitivity. It can realize simultaneous detection of 12 common gastrointestinal pathogens on the same detection platform. The detection can be completed within 50 minutes at the fastest, realizing rapid detection of 12 common gastrointestinal pathogens, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown are the PCR amplification curves of three primers and probes targeting VC.

[0039] Figure 2 Shown are the PCR amplification curves of three primers and probes targeting VP.

[0040] Figure 3 Shown are the PCR amplification curves of three primers and probes targeting SE.

[0041] Figure 4 Shown are the PCR amplification curves of three primers and probes targeting SC.

[0042] Figure 5 Shown are the PCR amplification curves of three primers and probe targeting DEC.

[0043] Figure 6 Shown are the PCR amplification curves of three primers and probes targeting CA.

[0044] Figure 7Shown are the PCR amplification curves of three primers and probes targeting YE.

[0045] Figure 8 Shown are the PCR amplification curves of three primers and probes targeting CD.

[0046] Fig. 9 Shown are the PCR amplification curves of three primers and probes targeting ES.

[0047] Fig.10 PCR amplification curves of three primers and probes targeting AH.

[0048] Fig.11 Shown are the PCR amplification curves of three primers and probes targeting PS.

[0049] Fig.12 : PCR amplification curves of three primers and probes targeting VF.

[0050] Fig.13 Shown are the PCR amplification curves of combination 1 with different concentrations of VC, VP, and SE.

[0051] Fig.14 PCR amplification curves of combination 2 with different concentrations of SC, DEC, and CA.

[0052] Fig.15 PCR amplification curves of combination 3 with different concentrations of YE, CD, and ES.

[0053] Fig.16 PCR amplification curves of combination 4 with different concentrations of AH, PS, and VF.

[0054] Fig.17 PCR amplification curves of combination 5 with different concentrations of SE, SC, and DEC.

[0055] Fig.18 PCR amplification curves of combination 6 with different concentrations of SE, CA, and YE.

[0056] Fig.19 Shown are the PCR amplification curves of combination 7 with different concentrations of SE, CD, and ES.

[0057] Fig. 20 PCR amplification curves of combination 8 with different concentrations of SE, AH, and PS.

[0058] Fig.21 PCR amplification curve of specific test samples. DETAILED DESCRIPTION

[0059] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0060] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0061] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0062] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0063] The present invention is further described in detail below with reference to specific embodiments.

[0064] According to the relevant gastrointestinal pathogen databases and clinical sample sequencing analysis at home and abroad, 12 highly conserved region sequences of gastrointestinal pathogens were selected. Based on the design principles of primers and probes for fluorescent PCR, sequence analysis was performed on the NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore) and NUPACK https: / / www.nupack.org / analysis / input websites, and then a large number of primers and probes were designed. The following primers and probes are used as examples to illustrate the detection performance of primers and probes:

[0065] Primer and probe sequences for detecting 12 common gastrointestinal pathogens:

[0066] 1. Vibrio cholerae (VC)

[0067] Primer and probe 1:

[0068]

[0069] Primer and probe 2:

[0070]

[0071] Primer and probe 3:

[0072]

[0073] 2. Vibrio Parahaemolyticus (VP)

[0074] Primer and probe 1:

[0075]

[0076] Primer and probe 2:

[0077]

[0078] Primer and probe 3:

[0079]

[0080] 3. Salmonella enteriditis (SE)

[0081] Primer and probe 1:

[0082]

[0083] Primer and probe 2:

[0084]

[0085] Primer and probe 3:

[0086]

[0087] 4. Shigella Castellani (SC)

[0088] Primer and probe 1:

[0089]

[0090] Primer and probe 2:

[0091]

[0092] Primer and probe 3:

[0093]

[0094] 5. Diarrhea Escherichia coli (DEC)

[0095] Primer and probe 1:

[0096]

[0097] Primer and probe 2:

[0098]

[0099] Primer and probe 3:

[0100]

[0101]

[0102] 6. Campylobacter (CA)

[0103] Primer and probe 1:

[0104]

[0105] Primer and probe 2:

[0106]

[0107] Primer and probe 3:

[0108]

[0109] 7. Yersinia enterocolitica (YE)

[0110] Primer and probe 1:

[0111]

[0112] Primer and probe 2:

[0113]

[0114] Primer and probe 3:

[0115]

[0116] 8. Clostridioides difficile (CD)

[0117] Primer and probe 1:

[0118]

[0119] Primer and probe 2:

[0120]

[0121] Primer and probe 3:

[0122]

[0123] 9. Enterobacter sakazakii (ES)

[0124] Primer and probe 1:

[0125]

[0126]

[0127] Primer and probe 2:

[0128]

[0129] Primer and probe 3:

[0130]

[0131] 10. Aeromonas hydrophila (AH)

[0132] Primer and probe 1:

[0133]

[0134] Primer and probe 2:

[0135]

[0136] Primer and probe 3:

[0137]

[0138] 11. Plesiomonas Shigelloides (PS)

[0139] Primer and probe 1:

[0140]

[0141] Primer and probe 2:

[0142]

[0143] Primer and probe 3:

[0144]

[0145] 12. Vibrio fluvialis (VF)

[0146] Primer and probe 1:

[0147]

[0148] Primer and probe 2:

[0149]

[0150]

[0151] Primer and probe 3:

[0152]

[0153] 13. Internal standard gene

[0154] MS2 bacteriophage gene fragment.

[0155] Primers and probes:

[0156]

[0157] The method for detecting 12 gastrointestinal pathogens is as follows:

[0158] The PCR reaction solution was prepared, including: Tris-HCl (pH 7.4), 10% Tween-20, 1 mg / mL BSA, 1M MgCl2, 5% DMSO, 0.04 U / μL DNA polymerase, 0.04 U / μL UNG enzyme, primers and probes.

[0159] The PCR reaction solution is placed in a fluorescent quantitative PCR instrument for reaction; after the reaction is completed, the 12 gastrointestinal pathogens are identified by the amplification curve or CT value.

[0160] The forward primers F used to detect the 12 gastrointestinal pathogens are used at a concentration of 150nM to 500nM, preferably 150nM to 300nM, and more preferably 150nM to 200nM; the reverse primers R are used at a concentration of 150nM to 500nM, preferably 150nM to 300nM, and more preferably 150nM to 200nM; the probes P are used at a concentration of 125nM to 300nM, preferably 125nM to 200nM, and more preferably 125nM to 150nM.

[0161] The CT value analysis and judgment criteria for single or multiplex PCR detection using the above primers and probes are shown in Table 1:

[0162] Table 1

[0163] Target CT value interpretation result VC ≤40 Vibrio cholerae VP ≤40 Vibrio parahaemolyticus SE ≤40 salmonella SC ≤40 Shigella DEC ≤40 Diarrheagenic Escherichia coli CA ≤40 Campylobacter YE ≤40 Yersinia enterocolitica CD ≤40 Clostridium difficile ES ≤40 Cronobacter sakazakii AH ≤40 Aeromonas hydrophila PS ≤40 Pleistomonas shigelloides VF ≤40 Vibrio fluvibrios MS2 ≤32 -

[0164] Example 1 Primer and probe screening

[0165] Twelve gastrointestinal pathogenic bacteria nucleic acid samples were selected, the enzyme was selected from Zhuhai Baorui Biotechnology, the primers and probes were synthesized by Sangon Biotechnology, and the instrument was ABI's QuantStudio Q5 model fluorescent quantitative PCR instrument. The test results were judged by amplification curves or CT values.

[0166] 1. Universal samples (covering multiple types) used ATCC strains, GDMCC strains and clinical samples. The samples were extracted with MagPure Bacterial DNA Kit. The operation was carried out strictly according to the instructions. The sample information is shown in Table 2.

[0167] Table 2

[0168] Sample name source Strain number concentration Vibrio cholerae GDMCC 1.2719 10^4CFU / mL Vibrio parahaemolyticus ATCC ATCC 17802 10^4CFU / mL salmonella GDMCC 1.3177 10^4CFU / mL Shigella GDMCC 1.424 10 people 4CFU / mL Diarrheagenic Escherichia coli GDMCC 1.3834 10^4CFU / mL Campylobacter ATCC BMZ147187 10^4CFU / mL Yersinia enterocolitica GDMCC 186042 10^4CFU / mL Clostridium difficile GDMCC 1.2602 10^4CFU / mL Cronobacter sakazakii GDMCC 1.2778 10^4CFU / mL Aeromonas hydrophila GDMCC 1.551 10^4CFU / mL Pleistomonas shigelloides GDMCC 1.4567 10^4CFU / mL Vibrio fluvibrios GDMCC 1.488 10^4CFU / mL Negative clinical samples - -

[0169] 2. The PCR amplification system is prepared as shown in Table 3:

[0170] Table 3

[0171] Reagents Final concentration Tris-HCl, pH 7.4 100mM 10% Tween-20 0.5％ 1mg / mL BSA 2 μg / mL <![CDATA[1MMgCl2]]> 10mM dNTPs 0.5mM DNA polymerase 0.04U / μL UNG enzyme 0.04U / μL DMSO 5％ Forward primer 200nM Reverse primer 200nM Probe 125nM

[0172] 3. The PCR amplification procedure is as shown in Table 4:

[0173] Table 4

[0174]

[0175] 4. Results Analysis

[0176] Amplification curve results for VC, VP, SE, SC, DEC, CA, YE, CD, ES, AH, PS, and VF are shown in Figures 1 to 12 , the test results (CT value) are shown in Table 5 below:

[0177] Table 5

[0178]

[0179]

[0180] The detection concentration of the sample in this embodiment is 10^4CFU / mL, and only the primer and probe set with higher sensitivity can achieve detection. According to the results in the above table, the primer and probe 1 of Vibrio cholerae, the primer and probe 1 of Vibrio parahaemolyticus, the primer and probe 3 of Salmonella, the primer and probe 2 of Shigella, the primer and probe 1 of diarrheagenic Escherichia coli, the primer and probe 2 of Campylobacter, the primer and probe 1 of Yersinia enterocolitica, the primer and probe 3 of Clostridium difficile, the primer and probe 2 of Cronobacter sakazakii, the primer and probe 1 of Aeromonas hydrophila, the primer and probe 2 of Shigella-like Pseudomonas, and the primer and probe 3 of Vibrio rivulosa can effectively detect the corresponding samples, while other subtypes and negative clinical samples cannot be detected, indicating that the above 12 gastrointestinal pathogen primer and probe sets have high sensitivity and good specificity.

[0181] Example 2 Multiple System Sensitivity Test

[0182] This example tests the multiplex PCR amplification effects of the 12 primers and probes screened in Example 1.

[0183] Twelve gastrointestinal pathogenic bacteria nucleic acid samples were selected, the enzyme was selected from Zhuhai Baorui Bio, the primer probe was synthesized by Sangon Biotechnology, and the instrument was ABI's QuantStudio Q5 model fluorescent quantitative PCR instrument. The test results were interpreted and judged by amplification curves or CT values.

[0184] Due to the limitation of the fluorescence channel of the detection instrument (only 4 fluorescence channels), each multiplex PCR system can only contain a combination of primers and probes for 2 to 3 gastrointestinal pathogens and 1 internal standard gene. The primers and probes for the 12 gastrointestinal pathogens of the present invention can be arbitrarily combined and can effectively achieve detection. The following are some of the combinations listed: Combination 1: VC, VP, SE and MS2; Combination 2: SC, DEC, CA and MS2; Combination 3: YE, CD, ES and MS2; Combination 4: AH, PS, VF and MS2; Combination 5: SE, SC, DEC and MS2; Combination 6: SE, CA, YE and MS2; Combination 7: SE, CD, ES and MS2; Combination 8: SE, AH, PS and MS2.

[0185] 1. The samples used were GDMCC strains, ATCC strains and clinical samples. The samples were extracted with TIANamp Bacteria DNA Kit for nucleic acid. The operation was carried out strictly in accordance with the instructions. The sample information is shown in Table 6.

[0186] Table 6

[0187]

[0188]

[0189] 2. The PCR amplification system was prepared as shown in Table 7. In the reaction systems of combinations 1 to 8, the final concentrations of the forward primers for different gastrointestinal pathogens were 200 nM, the final concentrations of the reverse primers were 200 nM, and the final concentrations of the probes were 125 nM.

[0190] Table 7

[0191] Reagents Final concentration Tris-HCl, pH 7.4 100mM 10% Tween-20 0.5％ 1mg / mL BSA 4 μg / mL <![CDATA[1M MgCl2]]> 10mM dNTPs 0.5mM DNA polymerase 0.06U / μL UNG enzyme 0.04U / μL DMSO 8％ Forward primer 200nM Reverse primer 200nM Probe 125nM

[0192] 3. PCR amplification procedure is the same as Table 4.

[0193] 4. Results Analysis

[0194] Amplification curve results are shown in Figures 13 to 20 (Combination 1 is Fig.13 , combination 2 is Fig.14 , combination 3 is Fig.15 , combination 4 is Fig.16 , combination 5 is Fig.17 , combination 6 is Fig.18 , combination 7 is Fig.19 , combination 8 is Fig. 20 ), the test results are as follows Table 8-9: Table 8

[0195]

[0196] Table 9

[0197]

[0198] According to the results in the table above, the sensitivity of VC in combination 1 can reach 250CFU / mL, the sensitivity of VP can reach 250CFU / mL, and the sensitivity of SE can reach 500CFU / mL; the sensitivity of SC in combination 2 can reach 250CFU / mL, the sensitivity of DEC can reach 250CFU / mL, and the sensitivity of CA can reach 500CFU / mL; the sensitivity of YE in combination 3 can reach 250CFU / mL, the sensitivity of CD can reach 500CFU / mL, and the sensitivity of ES can reach 500CFU / mL; the sensitivity of AH in combination 4 can reach 250CFU / mL, the sensitivity of PS can reach 500CFU / mL, and the sensitivity of VF can reach 250CFU / mL; in combination 5, combination 6, combination 7 and combination 8, the detection sensitivity of various pathogens can reach 250CFU / mL. The sensitivity of the common gastrointestinal multiple detection kits on the market is above 1000CFU / mL, so the multiple PCR amplification using the primer and probe combination of the present invention has a higher sensitivity. In addition, negative clinical samples were not detected.

[0199] Example 3 Multiple system specificity test

[0200] In order to test the specificity of the primer and probe combination for detecting 12 gastrointestinal pathogens of the present invention, strains with high homology to gastrointestinal pathogens, other pathogens and clinical samples that are prone to cause gastrointestinal clinical symptoms and complications were selected as specific test samples. Virus samples were extracted with the TaKaRa MiniBEST Viral RNA / DNAExtractionKit Ver.5.0 kit, and bacterial samples were extracted with the TIANamp Bacteria DNAKit kit. The operation was strictly carried out in accordance with the instructions. The enzyme was selected from Zhuhai Baorui Bio, the primers and probes were synthesized at Sangon Biotech, and the instrument was ABI's QuantStudio Q5 model fluorescent quantitative PCR instrument. The test results were judged by amplification curves or CT values.

[0201] The primer and probe combinations for detecting 12 gastrointestinal pathogens are exemplified by combinations 1 to 4 in Example 2, namely, combination 1: VC, VP, SE and MS2; combination 2: SC, DEC, CA and MS2; combination 3: YE, CD, ES and MS2; combination 4: AH, PS, VF and MS2.

[0202] 1. Sample information

[0203] The sample information is shown in Table 10.

[0204] Table 10

[0205]

[0206]

[0207] 2. The PCR amplification system was prepared as in Table 7. In the reaction systems of combinations 1 to 4, the final concentrations of the forward primers for different gastrointestinal pathogens were 200 nM, the final concentrations of the reverse primers were 200 nM, and the final concentrations of the probes were 125 nM.

[0208] 3. PCR amplification procedure is the same as Table 4.

[0209] 4. Results Analysis

[0210] PCR amplification curve results are shown in Fig.21 The test results are shown in Table 11:

[0211] Table 11

[0212]

[0213]

[0214] According to the results in the table above, norovirus, rotavirus, human enterovirus, astrovirus, Vibrio vulnificus, Bordetella pertussis, Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, fecal negative clinical samples and vomitus negative clinical samples all had no CT values, indicating that the primer and probe combination of this method has good specificity, and no strains with high homology to the 12 gastrointestinal pathogenic bacteria, other pathogens that are prone to cause gastrointestinal clinical symptoms and complications were detected. In addition, 4 positive samples of Salmonella, diarrhea-causing Escherichia coli, Vibrio parahaemolyticus and Clostridium difficile collected in clinical hospitals were all tested positive.

[0215] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer and probe combination for detecting gastrointestinal pathogens, characterized in that: The primer and probe combination includes at least two of the following primers and probes for 12 gastrointestinal pathogens: Primers and probes for Vibrio cholerae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.1, the reverse primer shown in SEQ ID NO.2, and the probe shown in SEQ ID NO.3; Primers and probes for Vibrio parahaemolyticus, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.10, the reverse primer shown in SEQ ID NO.11, and the probe shown in SEQ ID NO.12; Primers and probes for Salmonella, whose nucleotide sequences are the forward primer shown in SEQ ID NO.25, the reverse primer shown in SEQ ID NO.26, and the probe shown in SEQ ID NO.27; Primers and probes for Shigella, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.31, the reverse primer shown in SEQ ID NO.32, and the probe shown in SEQ ID NO.33; Primers and probes for diarrheagenic Escherichia coli, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.37, the reverse primer shown in SEQ ID NO.38, and the probe shown in SEQ ID NO.39; Primers and probes for Campylobacter, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.49, the reverse primer shown in SEQ ID NO.50, and the probe shown in SEQ ID NO.51; Primers and probes for Yersinia enterocolitica, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.55, the reverse primer shown in SEQ ID NO.56, and the probe shown in SEQ ID NO.57; The primers and probes for Clostridium difficile have nucleotide sequences as shown in SEQ ID NO.70 for the forward primer, SEQ ID NO.71 for the reverse primer, and SEQ ID NO.72 for the probe: Primers and probes for Cronobacter sakazakii, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.76, the reverse primer shown in SEQ ID NO.77, and the probe shown in SEQ ID NO.78; Primers and probes for Aeromonas hydrophila, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.82, the reverse primer shown in SEQ ID NO.83, and the probe shown in SEQ ID NO.84; Primers and probes for Pleistomonas shigellae, the nucleotide sequences of which are the forward primer shown in SEQ ID NO.94, the reverse primer shown in SEQ ID NO.95 and the probe shown in SEQ ID NO.96; The primers and probes for Vibrio fluvibrio have nucleotide sequences as shown in SEQ ID NO.106 for the forward primer, SEQ ID NO.107 for the reverse primer and SEQ ID NO.108 for the probe.

2. The primer and probe combination for detecting gastrointestinal pathogens according to claim 1, characterized in that: The primer and probe combination includes: primers and probes for the Salmonella, and primers and probes selected from at least one of Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like bacteria, and Vibrio fluvii.

3. The primer and probe combination for detecting gastrointestinal pathogens according to claim 2, characterized in that: The primer and probe combination includes the primers and probes for the Salmonella and the Shigella and diarrheagenic Escherichia coli; or, The primers and probes for the Salmonella and Campylobacter and Yersinia enterocolitica; or, The primers and probes for the Salmonella, Clostridium difficile and Cronobacter sakazakii; or, The primers and probes for the Salmonella and for Aeromonas hydrophila and Shigella oxytoca.

4. The primer and probe combination for detecting gastrointestinal pathogens according to claim 2, characterized in that: The primer and probe combination includes the primers and probes for the Salmonella and for Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like Pseudomonas and Vibrio fluvii.

5. The primer and probe combination for detecting gastrointestinal pathogens according to claim 1, characterized in that: The primer and probe combination also includes primers and probes for an internal standard gene; preferably, the internal standard gene is an MS2 phage gene fragment, and the primers and probes for the internal standard gene are nucleotide sequences such as a forward primer shown in SEQ ID NO.109, a reverse primer shown in SEQ ID NO.110, and a probe shown in SEQ ID NO.

111.

6. A kit for detecting gastrointestinal pathogens, characterized in that: The method comprises the primer and probe combination according to any one of claims 1 to 5.

7. A method for detecting gastrointestinal pathogens, characterized in that: The following steps are involved: Take DNA of the sample to be tested, and perform multiplex PCR amplification using the primer and probe combination described in any one of claims 1 to 5, or the kit described in claim 6.

8. The detection method according to claim 7, characterized in that The reaction system of the multiplex PCR amplification comprises the following components: Tris-HCl, Tween-20, BSA, MgCl2, dNTPs, DNA polymerase, UNG enzyme, the primer and probe combination.

9. The detection method according to claim 8, characterized in that: The concentration of the primers for Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Pseudomonas shigellae and Vibrio fluvii in the reaction system is 150nM to 500nM, preferably 150nM to 300nM, more preferably 150nM to 200nM; and / or, The concentrations of the probes for Salmonella, Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheagenic Escherichia coli, Campylobacter, Yersinia enterocolitica, Clostridium difficile, Cronobacter sakazakii, Aeromonas hydrophila, Shigella-like bacteria and Vibrio fluvii in the reaction system are each 125nM to 300nM, preferably 125nM to 200nM, and more preferably 125nM to 150nM.

10. The detection method according to any one of claims 7 to 9, characterized in that: The reaction procedure of the multiplex PCR amplification is: 1) 95° C., 60 s; 2) 95° C., 5 s; 60° C.; 25 s; 40 cycles.

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