Primer combination and kit for detecting intestinal infection pathogens and application

Abstract

The invention discloses a primer combination and a kit for detecting intestinal infection pathogens and application. The primer combination for detecting pathogens disclosed by the invention comprises or consists of the following components: 28 amplification primers and 14 quality probe extension primers. 13 pathogens can be detected at a time, the detection cost is low, the operation is simple and convenient, and the pathogen detection efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a primer combination and a kit and application thereof for detecting intestinal infection pathogens, especially intestinal infection pathogens of experimental animals (such as mice and rats). Background Art

[0002] At present, the standard methodologies for identifying pathogenic microorganisms in the intestines of mice and rats are culture method and ELISA antibody detection method, and ordinary PCR method is commonly used in scientific research.

[0003] As far as the culture method is concerned, it is the gold standard for detecting pathogenic microorganisms, but the positive rate is low, the inspection cycle is long, and it is difficult to detect pathogens that are difficult to culture, which often leads to missed detections.

[0004] For the detection of viral infections and difficult-to-culture pathogens (such as Tyzer pathogens), ELISA is often used in experimental animal testing to detect antiviral serum antibodies or pathogen serum antibodies. This method is simple to operate, based on the binding of antigen and antibody, and has a certain specificity and sensitivity. However, the ELISA methodology, whether manual or instrumental, is greatly affected by temperature, time, etc., and there is a time window for the production of antibodies. Low serum antibody levels in the early or late stages of infection may result in missed detections, and the detection throughput is low.

[0005] The real-time fluorescence quantitative PCR (Quantitative Real-time polymerase chain reaction, qPCR) methodology can improve the sensitivity of detection to a certain extent and make up for the shortcomings of the aforementioned methodology. However, the current qPCR methodology has limited targets for one-time detection. Due to the limitation of the fluorescence channel of the PCR detection instrument, it can detect up to 4 targets at one time. For the detection of more than 4 targets, it needs to be divided into multiple wells and multiple detections, which is more complicated to operate and has high labor and reagent costs. Although there are also developments based on PCR methodology, such as microfluidic chip method and constant temperature amplification method, which can detect multiple targets, the sensitivity is lacking, and there is currently no kit for the detection of intestinal pathogens in experimental animals. Summary of the invention

[0006] Experimental animals refer to animals that are artificially bred, have controlled microorganisms, have a clear genetic background or origin, and are used for scientific research, teaching, production, testing and other scientific experiments.

[0007] Tyzzer's organism is a long filamentous bacillus that is difficult to culture artificially. When experimental animals are infected, it causes Tyzzer's disease, which is characterized by watery diarrhea, hemorrhagic necrotizing colitis and appendicitis, and multifocal liver necrosis. It is an important pathogen of concern in intestinal infections of experimental animals.

[0008] Salmonella is a gram-negative bacillus that can cause intestinal infections in a variety of animals, so salmonellosis is also called "paratyphoid fever" or "enteric disease." Salmonella typhimurium is prone to cause natural infection in experimental animals, and the infection is characterized by digestive tract diseases and acute diarrhea.

[0009] Corynebacterium bovis is a Gram-positive short rod-shaped bacillus and an important pathogen of bovine mastitis. In experimental animals, the bacteria mainly cause infection in immunodeficient mice, but most of them do not show clinical symptoms. It mainly causes hyperkeratosis in nude mice, causing hyperkeratotic dermatitis and acanthosis in nude mice. It often exists in the oral cavity and digestive tract. It is an important pathogen of intestinal infection in experimental animals. Feces is one of the main sources of specimens for daily monitoring. Once hairless mice and immunodeficient mice are infected, they carry the disease almost for life. Animal infection is usually accompanied by weight loss, increased water intake, conjunctival congestion, slow growth of xenografts, etc. Once the facilities are contaminated, it is difficult to remove.

[0010] Enterohemorrhagic Escherichia coli O157:H7 is an important zoonotic pathogen that can cause serious infectious diarrhea. It is usually transmitted through food and can infect livestock and poultry, as well as humans. It can cause diarrhea, hemorrhagic colitis, etc., and can be fatal in severe cases. The bacteria can cause severe diarrhea in experimental animals and pose a threat of secondary infection to experimental operators. Experimental studies on mouse models infected with enterohemorrhagic Escherichia coli O157:H7 have shown that infected model mice can cause intestinal edema, bleeding, kidney damage, liver damage, etc., but there are also mouse strains that are not sensitive to the pathogen. The bacteria is an important intestinal pathogen for microbial detection in experimental animals.

[0011] Minute virus of mice (MVM), Theiler'smurine encephalomyelitis virus, Mouse Hepatitis Virus, Kilham Rat Virus, and Toolan's H-1Virus are internationally popular infectious viruses for experimental mice and rats. Most of them are latent infections with no obvious clinical symptoms. However, studies have shown that in experiments involving the immune system, tumors, and transplantation, in vivo and in vitro viral infections seriously affect the accuracy and credibility of experimental data. Once infected, experimental animals will be in a state of carrying and excreting viruses for a long time. Contamination of feces, bedding, and feed will lead to the spread of the virus. Therefore, timely and comprehensive detection and rapid cutting off of the source of infection are very necessary for the quality control of experimental animals.

[0012] Murine Norovirus is the only Norovirus that can replicate in cell culture and small animal models. It can be used to construct small animal models to study the biology and pathogenesis of Norovirus. MNV is also an ideal virus model for studying human Norovirus. Murine Norovirus has a high infection rate in experimental mice, and all strains are susceptible. According to the survey, 67.3% of ordinary mice in Japan, 39.1% of SPF mice, and 62.5% of ordinary mice in the United States were infected with MNV, and 20% of commercial SPF C57BL / 6 mice were found to have MNV antibodies. Although mice with normal immune function have no obvious symptoms after infection, they can exist in the body for a long time and can excrete toxins in feces for a long time; immunodeficient mice infected with mouse Norovirus may cause obvious pathological damage, but there are also reports of no obvious symptoms. Since infected mice have the risk of disease transmission and have adverse effects on experimental results, they are routinely monitored pathogens in experimental mice. Studies have shown that the infection rate of MNV in laboratory mouse populations is high in normal barrier breeding environments.

[0013] Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae are all conditional pathogens of zoonotic diseases, and are pathogens that must be excluded from SPF-level experimental animals such as rats, mice, guinea pigs, hamsters, rabbits, etc. in my country. The three pathogens may cause respiratory and digestive tract diseases under conditions of low immune function and experimental stimulation in experimental animals, and severe infection may even cause animal death. The results of laboratory animal quality monitoring in recent years show that conditional pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae are often positive, and the health risks and biosafety hazards caused to laboratory animal practitioners and animal experiment operators should not be underestimated. All three are important monitoring indicators for fecal microbial detection of experimental animals.

[0014] In summary, Tyzer pathogen, Salmonella typhimurium, Corynebacterium bovis, enterohemorrhagic Escherichia coli O157:H7, mouse parvovirus MVM strain, mouse encephalomyelitis virus, mouse hepatitis virus, rat parvovirus KRV strain, rat parvovirus H-1 strain, and mouse norovirus are 10 intestinal-related infection-related pathogens of concern to experimental animals. Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae are three conditional pathogens that are common to humans and animals, and are important indicators for fecal microbial monitoring of experimental animals.

[0015] Therefore, based on the current detection needs and status of intestinal pathogens in experimental animals, in order to improve detection efficiency and save costs, the present invention uses multiplex PCR mass spectrometry to design primer combinations and kits for the one-time detection of the above 13 pathogens.

[0016] In the first aspect, the present invention provides a primer combination for detecting pathogens, which includes or consists of: 28 amplification primers and 14 quality probe extension primers, wherein the nucleotide sequences of the amplification primers are shown in SEQ ID NOs: 1-28, and the nucleotide sequences of the quality probe extension primers are shown in SEQ ID NOs: 29-42.

[0017] The two types of primers, the amplification primers and the mass probe extension primers of the present invention, are packaged separately. The individual primers in the amplification primers can be packaged separately or mixed, and the individual primers in the mass probe extension primers can be packaged separately or mixed.

[0018] In some embodiments, in the above primer composition, the pathogen is an intestinal infection pathogen;

[0019] Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

[0020] In the present invention, amplification primers of the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 and mass probe extension primers of the sequence shown in SEQ ID NO:29 are used to detect the presence of the pathogen of Tyze;

[0021] Amplification primers of the sequences shown in SEQ ID NO:3 and SEQ ID NO:4 and mass probe extension primers of the sequence shown in SEQ ID NO:30 are used to detect the presence of Salmonella typhimurium;

[0022] Amplification primers of the sequences shown in SEQ ID NO:5 and SEQ ID NO:6 and mass probe extension primers of the sequence shown in SEQ ID NO:31 are used to detect the presence of Corynebacterium bovis;

[0023] Amplification primers of the sequences shown in SEQ ID NO:7 and SEQ ID NO:8 and mass probe extension primers of the sequence shown in SEQ ID NO:32 are used to detect the presence of Staphylococcus aureus;

[0024] Amplification primers of the sequences shown in SEQ ID NO:9 and SEQ ID NO:10 and mass probe extension primers of the sequence shown in SEQ ID NO:33 are used to detect the presence of Pseudomonas aeruginosa;

[0025] Amplification primers of the sequences shown in SEQ ID NO:11 and SEQ ID NO:12 and mass probe extension primers of the sequence shown in SEQ ID NO:34 are used to detect the presence of Klebsiella pneumoniae;

[0026] Amplification primers of the sequences shown in SEQ ID NO:13 and SEQ ID NO:14 and mass probe extension primers of the sequence shown in SEQ ID NO:35 are used to detect the presence of enterohemorrhagic Escherichia coli O157:H7;

[0027] Amplification primers of the sequences shown in SEQ ID NO:15 and SEQ ID NO:16 and mass probe extension primers of the sequence shown in SEQ ID NO:36 are used to detect the presence of the MVM strain of mouse parvovirus;

[0028] Amplification primers of the sequences shown in SEQ ID NO:17 and SEQ ID NO:18 and mass probe extension primers of the sequence shown in SEQ ID NO:37 are used to detect the presence of murine encephalomyelitis virus;

[0029] Amplification primers of the sequences shown in SEQ ID NO:19 and SEQ ID NO:20 and mass probe extension primers of the sequence shown in SEQ ID NO:38, in combination with amplification primers of the sequences shown in SEQ ID NO:21 and SEQ ID NO:22 and mass probe extension primers of the sequence shown in SEQ ID NO:39, are used to detect the presence of mouse and rat hepatitis virus;

[0030] Amplification primers of the sequences shown in SEQ ID NO:23 and SEQ ID NO:24 and mass probe extension primers of the sequence shown in SEQ ID NO:40 are used to detect the presence of rat parvovirus KRV strain;

[0031] Amplification primers of the sequences shown in SEQ ID NO:25 and SEQ ID NO:26 and mass probe extension primers of the sequence shown in SEQ ID NO:41 are used to detect the presence of rat parvovirus H-1 strain;

[0032] Amplification primers of the sequences shown in SEQ ID NO:27 and SEQ ID NO:28 and mass probe extension primers of the sequence shown in SEQ ID NO:42 are used to detect the presence of mouse norovirus.

[0033] In a second aspect, the present invention provides a kit for detecting pathogens, comprising any of the primer combinations described above.

[0034] In some embodiments, the above kit further comprises RT-PCR reaction reagents, PCR reaction reagents, dephosphorylation reaction reagents and / or mass probe extension reaction reagents.

[0035] In some embodiments, in any of the above-described kits, the pathogen is an intestinal infection pathogen;

[0036] Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

[0037] In a third aspect, the present invention provides a method for detecting pathogens, which is not used for diagnosis and treatment of diseases, comprising the following steps:

[0038] (1) extracting the total nucleic acid of the sample to be tested, and subjecting a portion of the total nucleic acid to RT-PCR to obtain cDNA;

[0039] (2) using the mixture of total nucleic acid and cDNA obtained in step (1) (the mass ratio of total nucleic acid to cDNA can be 1:(0.1-10)) as a template and the above 28 amplification primers as primers to perform a multiplex PCR reaction;

[0040] (3) dephosphorylating the multiplex PCR reaction products obtained in step (2) using alkaline phosphatase;

[0041] (4) using the 14 mass probe extension primers mentioned above to perform a single base extension reaction on the dephosphorylated product obtained in step (3);

[0042] (5) The single base extension reaction product obtained in step (4) is subjected to resin desalting and purification, and the reaction product is detected by mass spectrometry, and the pathogen negative / positive result is determined based on the single base extension result.

[0043] In some embodiments, in the above method, the alkaline phosphatase is shrimp alkaline phosphatase (SAP).

[0044] In some embodiments, in any of the above methods, the mass spectrometry is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0045] In some embodiments, in any of the above methods, the pathogen negative / positive result determination rules are as follows:

[0046] If a specific peak spectrum is detected at the mass position of 5426Da, the pathogen is positive for Tyzer, otherwise it is negative;

[0047] If a specific peak spectrum is detected at the mass position of 7870Da, Salmonella typhimurium is positive, otherwise it is negative;

[0048] If a specific peak spectrum is detected at the mass position of 7230Da, the test result is positive for Corynebacterium bovis, otherwise it is negative;

[0049] If a specific peak spectrum is detected at the mass position of 6625Da, Staphylococcus aureus is positive, otherwise it is negative;

[0050] If a specific peak spectrum is detected at the mass position of 7037Da, Pseudomonas aeruginosa is positive, otherwise it is negative;

[0051] If a specific peak spectrum is detected at the mass position of 5292Da, Klebsiella pneumoniae is positive, otherwise it is negative;

[0052] If a specific peak spectrum is detected at the mass position of 6947Da, then enterohemorrhagic Escherichia coli O157:H7 is positive, otherwise it is negative;

[0053] If a specific peak spectrum is detected at the mass position of 5632Da, the mouse parvovirus MVM strain is positive, otherwise it is negative;

[0054] If a specific peak spectrum is detected at the mass position of 5803Da, the mouse encephalomyelitis virus is positive, otherwise it is negative;

[0055] If specific peak spectra are detected at both mass positions 5517Da and 5654Da, the mouse hepatitis virus is positive, otherwise it is negative;

[0056] If a specific peak spectrum is detected at the mass position of 7379Da, the rat parvovirus KRV strain is positive, otherwise it is negative;

[0057] If a specific peak spectrum is detected at the mass position of 7612Da, the rat parvovirus H-1 strain is positive, otherwise it is negative;

[0058] If a specific peak spectrum is detected at the mass position of 6013Da, mouse norovirus is positive, otherwise it is negative.

[0059] In some embodiments, in any of the above methods, the pathogen is an intestinal infection pathogen;

[0060] Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

[0061] In a fourth aspect, the present invention provides use of any of the above-described primer combinations or any of the above-described kits in the preparation of products for detecting pathogens.

[0062] In some embodiments, in the above application, the pathogen is an intestinal infection pathogen;

[0063] Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

[0064] The present invention is a new methodology based on the combination of multiplex PCR and mass spectrometry, focusing on 10 intestinal pathogens of concern in experimental animals (such as mice and rats) and 3 pathogens of concern in zoonotic infections. The detection sensitivity is equivalent to that of qPCR, but 13 pathogens can be detected at one time. The detection cost is low, the operation is simple, and the pathogen detection efficiency is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Schematic diagram of the process of combining multiplex PCR and MALDI-TOF MS to qualitatively detect target sequences of pathogens.

[0066] Figure 2 The results of the qualitative detection of 13 pathogens by multiplex PCR combined with MALDI-TOF MS are shown in Figure 1. A shows the detection of pathogens 1-5, B shows the detection of pathogens 6-9, and C shows the detection of pathogens 10-13. The left side shows the positive result, and the right side shows the negative control. Sterile saline was used as the negative control. DETAILED DESCRIPTION

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0068] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0069] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0070] The Ultra high-efficiency and high-sensitivity stool / swab universal extraction kit was purchased from Guangzhou Ruilai Biological Detection Co., Ltd., with the catalog number Ultra-A01.

[0071] The Ultra Screen MS mass spectrometry kit was purchased from Guangzhou Ruilai Biological Detection Co., Ltd., with the catalog number Ultra-MS-01.

[0072] Example 1 Qualitative detection of 13 intestinal pathogenic microorganisms in rat feces by multiplex PCR mass spectrometry

[0073] This experiment is based on the combination of multiplex PCR and MALDI-TOF MS (i.e., multiplex PCR mass spectrometry), which can qualitatively detect the target sequence of pathogens. Specifically, primers are designed according to the pathogen-specific target sequence, and the pathogen-specific target sequence is enriched by multiplex PCR amplification. Specific MPE (Mass Probe Extension) primers are designed for the multiplex PCR amplification product sequence of the target sequence. The target sequence is identified by extending a base of different mass at the 3' site of the MPE primer. The product solution is spotted on the matrix of the target plate to form a co-crystal with the matrix, and the pathogen detection results are obtained by MALDI ionization and time-of-flight mass spectrometer separation and detection.

[0074] The schematic diagram of the process of combining multiplex PCR and MALDI-TOF MS to qualitatively detect the target sequence of pathogens is shown in Figure 1 shown.

[0075] 1. Primer design

[0076] The NCBI Gene database was used to search the gene library of the target pathogen, and the specific gene target fragment of the pathogen was selected. The Primer-BLAST tool was used to design PCR primers (forward primer PCR-F and reverse primer PCR-R), and the MPE primers were designed by the gene locus analysis design system, as shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080]

[0081] 2. Detection steps

[0082] (1) Sampling of fecal samples, extraction of total nucleic acid and reverse transcription

[0083] Use sterile EP tubes to collect fresh feces from 10 experimental rats (purchased from Guangdong Medical Experimental Animal Center, batch number 20240718). Pay attention to aseptic operation and extract total fecal nucleic acid in time or freeze it temporarily and then extract total fecal nucleic acid in batches.

[0084] Ultra high-efficiency and high-sensitivity stool / swab universal extraction kit was used to extract total nucleic acid (including DNA and RNA) from stool. 5-10 μL of total nucleic acid was reverse transcribed into cDNA (Ultra high-efficiency reverse transcription reagent (purchased from Guangzhou Ruilai Biological Detection Co., Ltd., catalog number Ultra-B01) was used).

[0085] (2) Multiplex PCR

[0086] The total nucleic acid obtained in step (1) and the reverse transcribed cDNA (mixed at a mass ratio of 1:1) were used as templates for multiplex PCR.

[0087] The multiplex PCR reaction system was prepared according to the Ultra Screen MS mass spectrometry kit, as shown in Table 2.

[0088] Table 2 Single-hole reaction system

[0089]

[0090]

[0091] Multiplex PCR reaction program: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 15 s, annealing at 59°C for 30 s, extension at 72°C for 30 s, reaction for 45 cycles; extension at 60°C for 10 min; storage at 4°C.

[0092] (3) SAP (shrimp alkaline phosphatase) digestion

[0093] SAP digests the remaining dNTPs in the multiplex PCR reaction products to prevent interference with subsequent single-base extension reactions.

[0094] Place the reaction plate after the multiplex PCR in step (2) in a centrifuge and centrifuge at 2000 rpm for 1 min, prepare a SAP mixture (prepared according to the Ultra Screen MS mass spectrometry kit in the following proportions: 1.53 μL of enzyme-free water, 0.17 μL of SAP buffer and 0.30 μL of SAP enzyme), dispense 2 μL of the SAP mixture / well into each well, mix well, and then perform a SAP digestion reaction.

[0095] SAP digestion reaction procedure: 37℃ reaction for 40min; 85℃ reaction for 5min; 4℃ storage.

[0096] (4) Single base extension

[0097] The SAP digestion reaction solution was instantaneously centrifuged, and an extension Mix-MPE mixed solution was prepared (prepared according to the Ultra Screen MS mass spectrometry kit in the following proportions: 1 μL of E-ddNTP mix, 1.4 μL of MPE buffer, 0.6 μL of MPE enzyme, and 1 μL of MPE primer mixed solution (containing all MPE primers in Table 1, and the concentrations of 14 MPE primers for pathogens No. 1 to 13 (where No. 10 includes 10-1 and 10-2 in sequence) in the mixed solution are: 9.01, 12.95, 11.78, 10.77, 11.45, 8.83, 11.29, 9.29, 9.53, 9.13, 9.32, 12.04, 12.46, and 9.84 μM, respectively), and 4 μL of the extension Mix-MPE mixed solution / well was dispensed into each well and mixed, and then a single base extension reaction was performed.

[0098] Single base extension reaction program: 40 cycles (95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 52°C annealing for 5 s, 80°C extension for 5 s, reaction for 5 cycles); 72°C extension for 3 min; 4°C storage.

[0099] (5) On-machine testing

[0100] The single base extension reaction product resin was desalted and then spotted on a plate, and detected by MALDI-TOF mass spectrometry. Detector: electron multiplier tube; laser wavelength: 337nm, nitrogen or semiconductor laser; flight distance: 800mm; resolution: 16 Daltons.

[0101] (6) Result determination

[0102] Mass spectrometry analysis software was used to analyze whether the pathogenic gene was detected. The rules for determining the detection results are shown in Table 3.

[0103] Table 3 Judgment rules for the results of multiplex PCR mass spectrometry detection of 13 pathogens

[0104]

[0105]

[0106]

[0107] In Table 3, negative “ / ” indicates that there is no corresponding single-base extension product, that is, no specific peak spectrum is detected at the corresponding mass position.

[0108] The stool specimen test results are shown in Table 4 Multiplex PCR mass spectrometry test results.

[0109] The serum of the above 10 experimental animal rats or the remaining specimens of the same fecal specimen were used for ELISA serum testing or culture testing according to the national standard GB14922-2022 "Microbiology, Parasitology Grade and Monitoring of Laboratory Animals" and DB32 / T 2599-2013 "Detection of Enterohemorrhagic Escherichia coli O157:H7 in Livestock and Poultry". The test results are shown in Table 4 for the ELISA and culture test results.

[0110] Table 4 Comparison of rat feces test results

[0111]

[0112] Result analysis: The multiplex PCR mass spectrometry method of the present invention detected 13 pathogens, and the results were consistent with those of traditional methodologies (ELISA method, culture method).

[0113] Example 2 Qualitative detection of a mixture of 13 intestinal pathogenic microorganisms by multiplex PCR mass spectrometry

[0114] The positive quality control products (components of inactivated pathogens) obtained from Guangdong Provincial Clinical Laboratory Center were used for total nucleic acid extraction (Ultra High Efficiency and High Sensitivity Feces / Swab Universal Extraction Kit) of No. 1 Taizé pathogen, No. 2 Salmonella typhimurium, No. 3 Corynebacterium bovis and No. 7 enterohemorrhagic Escherichia coli O157:H7, respectively, as the nucleic acids of No. 1, No. 2, No. 3 and No. 7 positive samples; No. 4 Staphylococcus aureus, No. 5 Pseudomonas aeruginosa and No. 6 Klebsiella pneumoniae were extracted using the Microbiology Laboratory of Guangdong Provincial People's Hospital. The preserved strains were expanded and cultured for total nucleic acid extraction (Ultra high-efficiency and high-sensitivity feces / swab universal extraction kit) and used as nucleic acids for positive samples No. 4, No. 5 and No. 6, respectively; the mouse parvovirus MVM strain No. 8, mouse encephalomyelitis virus No. 9, mouse hepatitis virus No. 10, rat parvovirus KRV strain No. 11, rat parvovirus H-1 strain No. 12 and mouse norovirus No. 13 used the positive quality control products (components of inactivated viruses) of Guangdong Provincial Clinical Laboratory Center for total nucleic acid extraction ( Ultra high-efficiency and high-sensitivity stool / swab universal extraction kit) was used, wherein the total nucleic acids of mouse parvovirus MVM strain 8, rat parvovirus KRV strain 11 and rat parvovirus H-1 strain 12 were used as positive sample nucleic acids of samples 8, 11 and 12, respectively, and the total nucleic acids of mouse encephalomyelitis virus 9, mouse hepatitis virus 10 and mouse norovirus 13 were reverse transcribed to obtain cDNA (Ultra high-efficiency reverse transcription reagent (purchased from Guangzhou Ruilai Biological Detection Co., Ltd., catalog number Ult ra-B01), and the obtained cDNA was used as No. 9, No. 10 and No. 13 positive sample nucleic acid respectively; No. 1-13 positive sample nucleic acid was diluted to 1pg / μL with sterile physiological saline, and then 1μL of each was taken for mixing to obtain a mixed solution, and then 1μL of the mixed solution was taken as the DNA template in Table 2, and multiple PCR, SAP (shrimp alkaline phosphatase) digestion, single base extension, machine detection and result determination were performed according to Example 1, and the DNA template was replaced with sterile physiological saline as a negative control.

[0115] Results of multiplex PCR mass spectrometry for detecting 13 pathogens at one time Figure 2 As shown; wherein, A shows the detection images of pathogens No. 1-5, B shows the detection images of pathogens No. 6-9, and C shows the detection images of pathogens No. 10-13.

[0116] Result analysis: In this experiment, multiplex PCR mass spectrometry can detect 13 pathogens at one time. By analyzing the positive samples and negative controls, it can be seen that there is no interference between the primers of the 13 pathogens. The positive samples all have specific peak spectra detected at the corresponding mass positions, while the negative controls have no peak spectra at the corresponding positions.

[0117] Example 3 Comparison of sensitivity of multiplex PCR mass spectrometry and isothermal amplification for detecting multiple pathogens

[0118] Purpose of the experiment: To compare the detection sensitivity of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa with the existing clinical one-time detection kits for multiple pathogens.

[0119] The instruments and test kits for respiratory pathogen nucleic acid detection (constant temperature amplification) 13 items (Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Haemophilus influenzae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, Methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis complex) were purchased from China Resources (Guangzhou) Medical Devices Co., Ltd. Testing instruments: RTisochip TM -A Constant temperature amplification microfluidic chip nucleic acid analyzer, detection methodology: microfluidic technology and constant temperature amplification technology. Common pathogens are shown in Table 5.

[0120] Table 5 Comparison of common pathogens with 13 respiratory pathogen nucleic acid detection (constant temperature amplification) kits

[0121]

[0122]

[0123] Experimental scheme: Rat feces were taken, and bacteria of a certain CFU concentration (Colony-Forming Units (CFU) refers to the total number of microorganisms such as bacteria, molds, yeasts, etc. in a unit volume) were mixed into the rat feces to prepare simulated specimens. Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa bacterial liquids were obtained from the amplified culture bacterial liquids of strains preserved in the Microbiology Laboratory of Guangdong Provincial People's Hospital. The concentrations were determined before use, and then total nucleic acid extraction was performed according to (1) in the detection step of Example 1. The mixing scheme and the quality of nucleic acid extraction are shown in Table 6.

[0124] Table 6 Simulated specimen preparation and nucleic acid extraction

[0125]

[0126] Each simulated specimen was tested according to the detection steps of Example 1 (ie, multiplex PCR mass spectrometry) and the isothermal amplification method. The results are shown in Table 7.

[0127] Table 7 Comparison and summary of the detection results of simulated specimens

[0128]

[0129]

[0130] For the one-time detection of Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa, this example compares the multiplex PCR mass spectrometry and the isothermal amplification method. The results show that the multiplex PCR mass spectrometry of the present invention is 1000 times more sensitive than the isothermal amplification method in detecting Staphylococcus aureus and 100 times more sensitive in detecting Pseudomonas aeruginosa. Although the sensitivity of detecting Klebsiella pneumoniae is the same, the multiplex PCR mass spectrometry of the present invention is more sensitive overall.

[0131] Example 4 Comparison of Multiplex PCR Mass Spectrometry and PCR for Detection of 13 Pathogens

[0132] Use sterile saline to dilute the positive sample nucleic acids No. 1-13 of Example 2 to 5 nucleic acid concentrations of 1ng / μL, 1pg / μL, 0.1pg / μL, 1fg / μL, and 0.1fg / μL. Use the following two detection schemes: A: Take 1μL of 13 positive sample nucleic acids of the same concentration and mix them to obtain a mixed solution, then take 1μL of the mixed solution as the DNA template in Table 2, and use the multiple PCR mass spectrometry of Example 1 for one-time detection; B: Use qPCR method to amplify each concentration of positive sample nucleic acid of each pathogen (qPCR detection reagent purchased from Guangzhou Ruilai Biological Detection Co., Ltd., item number Ultra-B02) (wherein the forward primer and reverse primer used are consistent with those in Table 1), and the PCR reaction procedure is: pre-denaturation 95℃30s; denaturation 95℃10s, annealing 60℃30s, 32 cycles. The test results are summarized in Table 8. The cost comparison of multiple PCR mass spectrometry for 13 targets and qPCR method is shown in Table 9.

[0133] Table 8 Summary of nucleic acid results of positive samples with different concentrations of pathogens 1-13 detected by two detection schemes

[0134]

[0135]

[0136] Table 9 Comparison of costs between multiplex PCR mass spectrometry for 13 targets and qPCR for single target

[0137]

[0138] By analyzing Table 8, it can be seen that the sensitivity of the multiplex PCR mass spectrometry method of the present invention in detecting 13 intestinal pathogen targets at one time is comparable to that of the qPCR method in detecting a single target. By analyzing Table 9, it can be seen that when detecting multiple targets, the multiplex PCR mass spectrometry method can significantly improve the detection efficiency, significantly reduce the difficulty of operation, reagent cost and time cost compared with the qPCR method.

Claims

1. A primer combination for detecting pathogens, comprising or consisting of: 28 amplification primers and 14 mass probe extension primers, wherein the nucleotide sequences of the amplification primers are shown in SEQ ID NOs: 1-28, and the nucleotide sequences of the mass probe extension primers are shown in SEQ ID NOs: 29-42.

2. The primer combination according to claim 1, characterized in that: The pathogen is an intestinal infection pathogen; Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

3. A kit for detecting pathogens, comprising the primer combination according to claim 1 or 2.

4. The kit according to claim 3, further comprising RT-PCR reaction reagents, PCR reaction reagents, dephosphorylation reaction reagents and / or mass probe extension reaction reagents.

5. The kit according to claim 3 or 4, characterized in that: The pathogen is an intestinal infection pathogen; Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

6. A method for detecting pathogens, which is not used for diagnosis and treatment of diseases, comprising the following steps: (1) extracting the total nucleic acid of the sample to be tested, and subjecting a portion of the total nucleic acid to RT-PCR to obtain cDNA; (2) using the mixture of total nucleic acid and cDNA obtained in step (1) as a template and the 28 amplification primers described in claim 1 as primers to perform a multiplex PCR reaction; (3) dephosphorylating the multiplex PCR reaction products obtained in step (2) using alkaline phosphatase; (4) using the 14 mass probe extension primers described in claim 1 to perform a single base extension reaction on the dephosphorylated product obtained in step (3); (5) The single base extension reaction product obtained in step (4) is subjected to resin desalting and purification, and the reaction product is detected by mass spectrometry, and the pathogen negative / positive result is determined based on the single base extension result.

7. The method according to claim 6, characterized in that: The alkaline phosphatase is shrimp alkaline phosphatase (SAP).

8. The method according to claim 6 or 7, characterized in that: The mass spectrometry adopts matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

9. The method according to any one of claims 6 to 8, characterized in that: The pathogen is an intestinal infection pathogen; Preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice MVM strain, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus, H-1), Murine Norovirus (MNV).

10. Use of the primer combination according to claim 1 or 2 or the kit according to any one of claims 3 to 5 in preparing a product for detecting pathogens; Preferably, the pathogen is an intestinal infection pathogen; More preferably, the intestinal infection pathogens include one or more of the following or consist of one or more of the following: Tyzzer's organism, Salmonella typhimurium, Corynebacterium bovis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, enterohemorrhagic Escherichia coli O157:H7, Minute virus of mice, Theiler's murine encephalomyelitis virus, Mouse Hepatitis Virus (MHV), Kilham Rat Virus (KRV), Toolan's H-1 Virus (H-1), Murine Norovirus (MNV).

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