Primer composition and kit for detecting infectious diarrhea pathogens

Abstract

The invention provides a primer composition and a kit for detecting infectious diarrhea type pathogens. Staphylococcus aureus, escherichia coli, klebsiella pneumoniae, proteus mirabilis, listeria monocytogenes, pseudomonas aeruginosa, salmonella enteritidis, clamping film bacillus aerogenes, vibrio cholerae, clostridium difficile, aeromonas capora, yersinia enterocolitica, plesiomonas shigelloides, vibrio parahaemolyticus, campylobacter jejuni, candida albicans and aspergillus fumigatus are detected at the same time. The kit can be one or any combination of Aspergillus flavus, entamoeba histolytica, human adenovirus A, human adenovirus B, human adenovirus C, human adenovirus F, human bocavirus, rotavirus, norovirus, fiveleaf virus and human astrovirus, and has the advantages of short time consumption, high resolution, high flux, high sensitivity and the like.

Description

Technical Field

[0001] The present invention relates to the field of pathogen detection, and in particular to a primer composition and a kit for detecting infectious diarrhea-type pathogens. Background Art

[0002] Infectious diarrhea is a common clinical disease, which can be caused by a variety of pathogens such as bacteria, viruses, fungi, protozoa and worms. People of different age groups are susceptible, showing the characteristics of group and high incidence. If infectious diarrhea is not effectively controlled and treated, once it develops into a severe disease, it will not only threaten the patient's life safety, but also may lead to the spread of pathogens and cause a wider range of infections. Therefore, the detection and research of diarrhea-type infections has a far-reaching impact on the prevention and control of diarrhea-type diseases.

[0003] The traditional diagnostic methods are mainly the following:

[0004] First, microscopic examination. Microscopic examination is a method for early diagnosis based on the morphological and structural characteristics of virus particles. It is particularly suitable for the diagnosis, classification and identification of viruses that cannot be propagated in in vitro cell culture or that can be cultured in vitro but require complex culture conditions. However, there are many disadvantages of using electron microscopy, such as being time-consuming and labor-intensive, and the diagnostic results largely rely on the operator's observation experience.

[0005] Second, cell culture. Using specific cells for cultivation and observing the effect of virus particles on cytopathic effects to detect viruses is another direct detection method. However, this method is cumbersome, laborious and time-consuming because the culture cycle of most viruses is as long as 4 to 8 days. In addition, this method has high requirements on the professional knowledge background and laboratory equipment of the operator. More importantly, many important enteroviruses cannot be detected by cell culture, or even if they can be cultured, the sensitivity of the neutralization between virus cells and antibodies is low, coupled with improper sample extraction, operation and handling, and cytopathic effects may not occur. Therefore, this method has certain limitations in its application.

[0006] Third, enzyme-linked immunosorbent assay. This test is to observe the immune complex formed by the enzyme-labeled antibody and the corresponding antigen after the substrate is colored, so as to judge the test result. However, since the specificity of the antigen-antibody reaction is not absolute, cross-reactions may occur in the ELISA test, which may lead to false positive test results. In addition, ELISA may not be able to effectively detect low concentrations of antigens or antibodies, especially in the early stages of infection when the concentration of pathogen antigens is low. The sensitivity of the test is not high enough and may lead to missed detections.

[0007] Fourth, molecular biology methods. With the application of molecular biology technology, microbiological detection technology has moved from biological examination to molecular biology identification. Different types of pathogenic microorganisms have specific gene sequences, so they can be detected by detecting the presence or absence of pathogenic microorganism-specific gene sequences. However, due to the similarity of the gene sequences of infectious diarrhea pathogens, the sensitivity of the molecular biology detection methods in the prior art is not high.

[0008] In other words, the current diagnostic methods for infectious diarrhea pathogens on the market have more or less disadvantages such as long time consumption, low resolution, low throughput, low sensitivity and labor-intensiveness. Summary of the invention

[0009] The purpose of the present invention is to provide a primer combination and a kit for detecting infectious diarrhea pathogens, aiming to overcome the problems of traditional detection methods such as long time consumption, low resolution, low throughput, low sensitivity and labor consumption, and achieve the effect of rapid and simultaneous detection of 28 infectious diarrhea pathogens.

[0010] To achieve the above objectives, the present technical solution provides a primer composition for detecting infectious diarrhea-type pathogens, comprising:

[0011] A Staphylococcus aureus primer set, comprising a first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 1 to 2, a second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 3 to 4, a third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 5 to 6, a fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 7 to 8, and a fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 9 to 10;

[0012] An Escherichia coli primer set, comprising a sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 11 to 12, a seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 13 to 14, an eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 15 to 16, a ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 17 to 18, and a tenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 19 to 20;

[0013] A Klebsiella pneumoniae primer set, comprising an eleventh primer pair having nucleotide sequences as shown in SEQ ID NOs: 21 to 22, a twelfth primer pair having nucleotide sequences as shown in SEQ ID NOs: 22 to 23, and a thirteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 24 to 25;

[0014] A Proteus mirabilis primer set, comprising a fourteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 26-27, a fifteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 28-29, and a sixteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 30-31;

[0015] A Listeria monocytogenes primer set, comprising a seventeenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 32-33, an eighteenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 34-35, a nineteenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 36-37, a twentieth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 39-40, and a twenty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 41-42;

[0016] A Pseudomonas aeruginosa primer set, comprising a 22nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 43-44, a 23rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 45-46, a 24th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 47-48, a 25th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 49-50, and a 26th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 51-52;

[0017] A Salmonella enterica primer set, comprising a 27th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 53-54, a 28th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 55-56, a 29th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 57-58, and a 30th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 59-60;

[0018] A primer set for Bacillus perfringens, comprising a 31st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 61-62, a 32nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 63-64, and a 33rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 65-66;

[0019] A Vibrio cholerae primer set, comprising a 34th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 67-68, a 35th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 69-70, a 36th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 71-72, and a 37th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 73-74;

[0020] A Clostridium difficile primer set, comprising a 38th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 75-76, a 39th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 77-78, a 40th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 79-80, a 41st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 81-82, and a 42nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 83-84;

[0021] A primer set for Aeromonas caviae, comprising a 43rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 85-86, a 44th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 87-88, a 45th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 89-90, a 46th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 91-92, and a 47th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 93-94;

[0022] A Yersinia enterocolitica primer set, comprising a 48th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 95-96, a 49th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 97-98, and a 50th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 99-100;

[0023] A primer set for Shigella-like Pleistomonas, comprising a fifty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 101 to 102, a fifty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 103 to 104, a fifty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 105 to 106, a fifty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 107 to 108, and a fifty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 109 to 110;

[0024] A Vibrio parahaemolyticus primer set, comprising a fifty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 111 to 112, a fifty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 113 to 114, a fifty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 115 to 116, a fifty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 117 to 118, and a sixtieth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 119 to 120;

[0025] A Campylobacter jejuni primer set, comprising a sixty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 121-122, a sixty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 123-124, a sixty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 125-126, and a sixty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 127-128;

[0026] A Candida albicans primer set, comprising a sixty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 129-130, a sixty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 131-132, a sixty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 133-134, a sixty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 135-136, a sixty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 137-138, and a seventyth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 139-140;

[0027] An Aspergillus fumigatus primer set, comprising a seventy-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 141-142, a seventy-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 143-144, a seventy-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 145-146, a seventy-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 147-148, and a seventy-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 149-150;

[0028] A primer set for Aspergillus flavus, comprising a seventy-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 151-152, a seventy-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 153-154, a seventy-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 155-156, a seventy-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 157-158, and an eightieth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 159-160;

[0029] An Entamoeba histolytica primer set, comprising an eighty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 161-162, an eighty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 163-164, an eighty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 165-166, and an eighty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 167-168;

[0030] A human adenovirus type A primer set, comprising an eighty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 169-170, an eighty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 171-172, an eighty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 173-174, an eighty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 175-176, an eighty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 177-178, a ninety-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 179-180, and a ninety-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 181-182;

[0031] A human adenovirus type B primer set, comprising a ninety-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 183-184, a ninety-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 185-186, a ninety-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 187-188, and a ninety-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 189-190;

[0032] A human adenovirus type C primer set, comprising a ninety-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 191 to 192, a ninety-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 193 to 194, a ninety-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 195 to 196, a ninety-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 197 to 198, and a hundredth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 199 to 200;

[0033] A human adenovirus F type primer set, comprising a 101st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 201-202, a 102nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 203-204, a 103rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 205-206, a 104th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 207-208, a 105th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 209-210, a 106th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 211-212, and a 107th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 213-214;

[0034] A human bocavirus primer set, comprising a 108th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 215-216 and a 109th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 217-218;

[0035] A rotavirus primer set, comprising a 110th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 219-220, a 111th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 221-222, and a 112th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 223-224;

[0036] A norovirus primer set, comprising a 113th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 225-226, a 114th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 227-228, a 115th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 229-230, and a 116th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 231-232;

[0037] Zaru virus primers, the Zaru virus primer set includes the 117th primer pair with nucleotide sequences as shown in SEQ ID NOs: 233-234, the 118th primer pair with nucleotide sequences as shown in SEQ ID NOs: 235-236, the 119th primer pair with nucleotide sequences as shown in SEQ ID NOs: 237-238, and the 200th primer pair with nucleotide sequences as shown in SEQ ID NOs: 239-240.

[0038] A human astrovirus primer set, comprising a 201st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 241-242.

[0039] The infectious diarrhea pathogen is one or any combination of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Clostridium perfringens, Vibrio cholerae, Clostridium difficile, Aeromonas caviae, Yersinia enterocolitica, Shigella-like bacteria, Vibrio parahaemolyticus, Campylobacter jejuni, Candida albicans, Aspergillus fumigatus, Aspergillus fumigatus, Entamoeba histolytica, human adenovirus type A, human adenovirus type B, human adenovirus type C, human adenovirus type F, human bocavirus, rotavirus, norovirus, zarovirus, and human astrovirus.

[0040] In some embodiments, each primer pair from the first primer pair to the second primer pair consists of a forward primer and a corresponding reverse primer. The first primer pair to the second primer pair of this scheme are selected from the reference genome of the corresponding pathogen, and after comparing the nt library, the kmer fragments with a coverage of more than 90% and a specificity of more than 90% are screened and designed.

[0041] In some embodiments, a Barocde sequence is added to each primer in each primer pair from the first primer pair to the two hundred and first primer pair, and the Barocde sequences of different infectious diarrhea-type pathogens are different. This has the advantage that when multiple infectious diarrhea-type pathogens are detected simultaneously in subsequent multiple PCR, different infectious diarrhea-type pathogens can be distinguished by identifying the Barocde sequence, so that multiple samples can be processed simultaneously in one experiment or multiple pathogens in the same sample can be detected, which greatly increases the detection throughput, can obtain more detection information in a short time, and meets the needs of large-scale detection.

[0042] In some embodiments, the 5' end of each primer in each primer pair from the first primer pair to the second hundred and first primer pair is phosphorylated. The advantage of this is that the 5' end phosphorylation modification can make the primer easier to react with molecules such as linkers in the ligation reaction, improve the ligation efficiency, ensure the smooth construction of the library, and thus provide high-quality templates for subsequent high-throughput sequencing; it can increase the chemical stability of the primer molecules, reduce the degradation of the primers during storage and experimental operations due to factors such as nucleases, extend the shelf life of the primers, and ensure that the primers can function stably during the experiment, thereby improving the repeatability and reliability of the experimental results.

[0043] The primer compositions for detecting infectious diarrhea-type pathogens are summarized in Table 1 below:

[0044] Table 1 Primer composition for detecting infectious diarrhea pathogens

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] In some embodiments, the present invention designs at least one primer pair for each diarrheal pathogen, which has the advantage of increasing the sensitivity and accuracy of detection.

[0052] In a second aspect, the present invention provides a kit for detecting infectious diarrhea-type pathogens, comprising the primer composition for detecting infectious diarrhea-type pathogens mentioned in the first aspect, an artificial plasmid set, and a multiplex polymerase chain reaction mixture.

[0053] In some embodiments, the artificial plasmid set of this scheme includes artificial plasmids of different infectious diarrhea-type pathogens, and the artificial plasmids have the same primer ends as the corresponding infectious diarrhea-type pathogen amplicons, there is an 18-22% difference in the middle region, and the GC content difference is 1% to 3%.

[0054] Preferably, the primer ends of the artificial plasmid and the corresponding infectious diarrhea pathogen amplicon are the same, there is a 20% difference in the middle region, and the GC content difference is 2%, which can ensure the efficiency of the same primer to simultaneously amplify the plasmid set and the target pathogen. Since the artificial plasmid set can consume a large number of primers, the generation of primer dimers can be further reduced. For negative samples, the artificial plasmid set can monitor the experimental process and avoid the problem of little data or no data in sequencing. Moreover, since the amplification efficiency of the artificial plasmid set is known, relative quantification can be achieved for the target sample.

[0055] In some embodiments, the artificial plasmid set includes one or any combination of an artificial plasmid of Staphylococcus aureus, an artificial plasmid of Escherichia coli, an artificial plasmid of Klebsiella pneumoniae, an artificial plasmid of Proteus mirabilis, an artificial plasmid of Listeria monocytogenes, an artificial plasmid of Pseudomonas aeruginosa, an artificial plasmid of Salmonella enterica, an artificial plasmid of Bacillus perfringens, an artificial plasmid of Vibrio cholerae, and an artificial plasmid of Clostridium difficile.

[0056] The nucleotide sequences of the artificial plasmids of each infectious diarrhea-type pathogen in the artificial plasmid set are shown in Table 2 below:

[0057] Table 2 Nucleotide sequences of artificial plasmid sets

[0058]

[0059]

[0060]

[0062] Correspondingly, the nucleotide sequence of the artificial plasmid of Staphylococcus aureus in this scheme is shown in SEQ ID NO.243, the nucleotide sequence of the artificial plasmid of Escherichia coli is shown in SEQ ID NO.244, the nucleotide sequence of the artificial plasmid of Klebsiella pneumoniae is shown in SEQ ID NO.245, the nucleotide sequence of the artificial plasmid of Proteus mirabilis is shown in SEQ ID NO.246, the nucleotide sequence of the artificial plasmid of Listeria monocytogenes is shown in SEQ ID NO.247, the nucleotide sequence of the artificial plasmid of Pseudomonas aeruginosa is shown in SEQ ID NO.248, the nucleotide sequence of the artificial plasmid of Salmonella enterica is shown in SEQ ID NO.249, the nucleotide sequence of the artificial plasmid of Bacillus perfringens is shown in SEQ ID NO.250, the nucleotide sequence of the artificial plasmid of Vibrio cholerae is shown in SEQ ID NO.251, and the nucleotide sequence of the artificial plasmid of Clostridium difficile is shown in SEQ ID NO.252.

[0063] In some embodiments, the primer composition for detecting infectious diarrhea-type pathogens, the artificial plasmid set, the multiple polymerase chain reaction mixture, the DNA template and the sample to be tested are mixed to perform multiple PCR amplification to obtain PCR products, wherein the amplification procedure is shown in Table 3 below:

[0064] Table 3 Amplification program

[0065]

[0066] In some embodiments, the PCR product is directly added to a sequencing adapter for sequencing, and the detection results of infectious diarrhea-type pathogens are obtained by sequencing.

[0067] In some embodiments, the method is applied to a nanopore sequencing platform for sequencing.

[0068] Compared with the prior art, this technical solution has the following characteristics and beneficial effects:

[0069] 1. This solution can simultaneously detect up to 28 infectious diarrhea pathogens such as Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. It covers multiple types of pathogens such as bacteria, viruses, fungi, and protozoa in one experiment, avoiding the tedious process of testing each pathogen separately, greatly improving the detection efficiency, reducing experimental time, reducing costs, reducing workload, and identifying pathogens of different types at the same time. It is especially suitable for large-scale clinical screening or diagnosis of complex infections. In combination with high-throughput sequencing technologies such as nanopore sequencing platforms, a large amount of sequence information can be quickly obtained, further improving the detection throughput, and being able to process a large number of samples in a short time to meet the needs of rapid detection of a large number of samples in scenarios such as public health monitoring.

[0070] 2. This scheme designs multiple pairs of specific primers for each pathogen. These primers are carefully selected from the corresponding pathogen reference genome, and are designed by screening out kmer fragments with a coverage of more than 90% and a specificity of more than 90% after comparison with the nt library. Different Barcode sequences are connected to the specific primers respectively, and the 5' end is phosphorylated at the same time. After one round of PCR, the sequencing adapter is directly added to the sequencing machine, and the pathogen database is compared to obtain pathogen detection results quickly and accurately, effectively avoiding false positive results caused by nonspecific amplification and cross-reaction, and ensuring the accuracy of the test results.

[0071] 3. The artificial plasmid set sequence designed in this scheme is the same as the target amplicon of the pathogen at the primer end, and there is a 20% difference in the middle region. In this way, the primers can amplify the relevant pathogens and plasmid sets at the same time. This method can improve the detection sensitivity, monitor the experimental process, and ensure the amount of experimental data and relative quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 It is a tape image of amplified strips of 17 infectious diarrhea-type pathogens.

[0073] Figures 2 to 5 It is the sequencing data of 28 infectious diarrhea-type pathogens. DETAILED DESCRIPTION

[0074] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0075] Embodiment 1

[0076] Specific primers were designed for diarrhea pathogens such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Capsella perfringens, Vibrio cholerae, Clostridium difficile, Aeromonas caviae, Yersinia enterocolitica, Shigella-like bacteria, Vibrio parahaemolyticus, Campylobacter jejuni, Candida albicans, Aspergillus fumigatus, Aspergillus fumigatus, Entamoeba histolytica, human adenovirus type A, human adenovirus type B, human adenovirus type C, human adenovirus type F, human bocavirus, rotavirus, norovirus, zaruvirus, and human astrovirus:

[0077] A 500bp kmer with a step size of 50bp was selected from the reference genome of the corresponding diarrheal pathogen, and then the nt library was compared to screen the kmer fragments with a coverage of more than 90% and a specificity of more than 90%. Primers were designed in batches using primer3, and dimer analysis was performed on the designed primers. Primers with a ΔG less than -10 were filtered to obtain a primer composition for detecting infectious diarrheal pathogens as shown in Table 1.

[0078] Specificity verification: One pair of primers was selected for each of the 17 pathogens: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Bacillus perfringens, Vibrio cholerae, Clostridium difficile, Aeromonas caviae, Yersinia enterocolitica, Pseudomonas shigellae, Vibrio parahaemolyticus, Campylobacter jejuni, Candida albicans, and Aspergillus fumigatus. The template concentration was 1*10 5 PCR amplification was performed at 1.5% agarose gel electrophoresis, and the PCR products were verified. The specificity was verified by 17 pathogenic bacterial culture samples, and the amplified band gel images of 17 bacterial culture samples were obtained as shown in the figure. Figure 1 As shown, experiments have proven that there is no cross-reaction between the two, the primer sequence interference competition is small, and the overall specificity is very good.

[0079] Embodiment 2:

[0080] (1) Sample pretreatment and nucleic acid extraction:

[0081] For the 17 bacterial culture samples in Example 1, the remaining 11 pathogens were selected as clinical samples, and the samples were extracted using QIAGEN's QIAampDNA Mini Kit (50) and QIAamp Viral RNA Mini Kit (250);

[0082] (2) Multiple target-specific responses

[0083] 10 μl of MultiplexPCR Mix at a concentration of 2X and 5 μl of primer combination for detecting infectious diarrhea pathogens, 5 μl of DNA template and artificial plasmid set were added respectively, and the PCR products were purified with 0.8x magnetic beads after equal mass pooling, wherein the primer combination for detecting infectious diarrhea pathogens is shown in Table 1, and each artificial plasmid in the artificial plasmid set is shown in Table 2, and each primer in the primer combination for detecting infectious diarrhea pathogens is equally divided, and each artificial plasmid in the artificial plasmid set is also equally divided. After mixing, amplification is performed according to the amplification program shown in Table 3 to obtain an amplified product.

[0084] (3) Connector ligation and purification:

[0085] 3.1 Configure the system according to the proportions shown in Table 4 below

[0086] Table 4 Configuration system ratio table

[0087]

[0088] 3.2 Place the PCR reaction tube on the PCR instrument, program: 24℃10min; 3.3 Transfer the PCR product to a new 1.5ml EP tube, add 80μl AMPure XP magnetic beads, mix by flicking the EP tube, and incubate at room temperature for 5 minutes; 3.4 Place the EP tube on the magnetic rack until the eluate is clear and colorless, then remove the supernatant; 3.5 Place the EP tube on the magnetic rack, wash the magnetic beads with 200μl SFB, remove the SFB and discard; 3.6 Repeat the previous step; 3.7 Centrifuge briefly and place the EP tube back on the magnetic rack to remove all residual ethanol; 3.8 Add 20μL EB eluate, remove the EP tube from the magnetic rack, and incubate at room temperature for 5 minutes; 3.9 Place the EP tube on the magnetic rack until the eluate is clear and colorless; 3.10 Take all the supernatant into a new 1.5ml EB tube; 3.11 Take 1μl of the purified product and use Qubit dsDNA HS Assay Kit detection concentration.

[0089] (4) Sequencing:

[0090] 4.1 Melt Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) at room temperature, vortex to mix, centrifuge briefly, and keep on ice for later use; 4.2 Add 30 μL of FCT to a tube of FCF, vortex to mix, and prepare a priming mixture; 4.3 Open the cover of the chip's cleaning port and remove bubbles as follows: A. Adjust the 1000 μL pipette to 200 μL; B. Gently insert the tip of the gun into the cleaning port and hold it perpendicular to the plane of the chip; C. Turn the pipette wheel until the scale shows 220-230 μL, or see a small volume of buffer enter the pipette tip; 4.4 Add 800 μL of the priming mixture to the chip cleaning port, avoid generating bubbles during the addition process, and let it stand for 5 minutes;

[0091] 4.5 During the static process, configure the library on the machine according to the proportion in Table 5 below:

[0092] Table 5 Computer library configuration table

[0093] Element volume Sequencing Buffer (SB) 37.5μl Library Beads (LIB) 25.5μl DNA library (100ng) 12μl total 75μl

[0094] 4.6 Add the remaining 200 μL of the initiation mixture to the chip cleaning port, and avoid introducing bubbles during the addition process; 4.7 Add 75 μL of the loading mixture to the chip dropwise through the chip injection port; 4.8 Close the chip injection port and chip cleaning port; 4.9 Load the chip into GridION / MinION for sequencing.

[0095] (5) Bioinformatics analysis;

[0096] Perform bioinformatics analysis on the offline data, compare it with the pathogen database, and analyze the test results to obtain the following results: Figures 2 to 5 shown.

[0097] Embodiment 3:

[0098] Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Capsella perfringens, Vibrio cholerae and Clostridium difficile were tested according to the steps of Example 2, and the corresponding control samples without adding artificial plasmids were designed for testing, and the test results were organized into Table 6:

[0099] Table 6 Test results

[0100]

[0101] From the results in Table VI, we can see that after adding the artificial plasmid set, the overall sensitivity is significantly improved.

[0102] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A primer composition for detecting infectious diarrhea-type pathogens, characterized in that: include: A Staphylococcus aureus primer set, comprising a first primer pair having nucleotide sequences as shown in SEQ ID NOs: 1 to 2, a second primer pair having nucleotide sequences as shown in SEQ ID NOs: 3 to 4, a third primer pair having nucleotide sequences as shown in SEQ ID NOs: 5 to 6, a fourth primer pair having nucleotide sequences as shown in SEQ ID NOs: 7 to 8, and a fifth primer pair having nucleotide sequences as shown in SEQ ID NOs: 9 to 10; An Escherichia coli primer set, comprising a sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 11 to 12, a seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 13 to 14, an eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 15 to 16, a ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 17 to 18, and a tenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 19 to 20; A Klebsiella pneumoniae primer set, comprising an eleventh primer pair having nucleotide sequences as shown in SEQ ID NOs: 21 to 22, a twelfth primer pair having nucleotide sequences as shown in SEQ ID NOs: 22 to 23, and a thirteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 24 to 25; A Proteus mirabilis primer set, comprising a fourteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 26-27, a fifteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 28-29, and a sixteenth primer pair having nucleotide sequences as shown in SEQ ID NOs: 30-31; A Listeria monocytogenes primer set, comprising a seventeenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 32-33, an eighteenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 34-35, a nineteenth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 36-37, a twentieth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 39-40, and a twenty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 41-42; A Pseudomonas aeruginosa primer set, comprising a 22nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 43-44, a 23rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 45-46, a 24th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 47-48, a 25th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 49-50, and a 26th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 51-52; A Salmonella enterica primer set, comprising a 27th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 53-54, a 28th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 55-56, a 29th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 57-58, and a 30th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 59-60; A primer set for Bacillus perfringens, comprising a 31st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 61-62, a 32nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 63-64, and a 33rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 65-66; A Vibrio cholerae primer set, comprising a 34th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 67-68, a 35th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 69-70, a 36th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 71-72, and a 37th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 73-74; A Clostridium difficile primer set, comprising a 38th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 75-76, a 39th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 77-78, a 40th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 79-80, a 41st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 81-82, and a 42nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 83-84; A primer set for Aeromonas caviae, comprising a 43rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 85-86, a 44th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 87-88, a 45th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 89-90, a 46th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 91-92, and a 47th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 93-94; A Yersinia enterocolitica primer set, comprising a 48th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 95-96, a 49th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 97-98, and a 50th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 99-100; A primer set for Pleistomonas shigellae, comprising a fifty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 101 to 102, a fifty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 103 to 104, a fifty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 105 to 106, a fifty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 107 to 108, and a fifty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 109 to 110; A Vibrio parahaemolyticus primer set, comprising a fifty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 111 to 112, a fifty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 113 to 114, a fifty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 115 to 116, a fifty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 117 to 118, and a sixtieth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 119 to 120; A Campylobacter jejuni primer set, comprising a sixty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 121-122, a sixty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 123-124, a sixty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 125-126, and a sixty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 127-128; A Candida albicans primer set, comprising a sixty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 129-130, a sixty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 131-132, a sixty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 133-134, a sixty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 135-136, a sixty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 137-138, and a seventyth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 139-140; An Aspergillus fumigatus primer set, comprising a seventy-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 141-142, a seventy-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 143-144, a seventy-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 145-146, a seventy-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 147-148, and a seventy-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 149-150; A primer set for Aspergillus flavus, comprising a 76th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 151-152, a 77th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 153-154, a 78th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 155-156, a 79th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 157-158, and an 80th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 159-160; An Entamoeba histolytica primer set, comprising an eighty-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 161-162, an eighty-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 163-164, an eighty-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 165-166, and an eighty-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 167-168; A human adenovirus type A primer set, comprising an eighty-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 169-170, an eighty-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 171-172, an eighty-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 173-174, an eighty-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 175-176, an eighty-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 177-178, a ninety-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 179-180, and a ninety-first primer pair having a nucleotide sequence as shown in SEQ ID NOs: 181-182; A human adenovirus type B primer set, comprising a ninety-second primer pair having a nucleotide sequence as shown in SEQ ID NOs: 183-184, a ninety-third primer pair having a nucleotide sequence as shown in SEQ ID NOs: 185-186, a ninety-fourth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 187-188, and a ninety-fifth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 189-190; A human adenovirus type C primer set, comprising a ninety-sixth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 191 to 192, a ninety-seventh primer pair having a nucleotide sequence as shown in SEQ ID NOs: 193 to 194, a ninety-eighth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 195 to 196, a ninety-ninth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 197 to 198, and a hundredth primer pair having a nucleotide sequence as shown in SEQ ID NOs: 199 to 200; A human adenovirus F type primer set, comprising a 101st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 201-202, a 102nd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 203-204, a 103rd primer pair having a nucleotide sequence as shown in SEQ ID NOs: 205-206, a 104th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 207-208, a 105th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 209-210, a 106th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 211-212, and a 107th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 213-214; A human bocavirus primer set, comprising a 108th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 215-216 and a 109th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 217-218; A rotavirus primer set, comprising a 110th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 219-220, a 111th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 221-222, and a 112th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 223-224; A norovirus primer set, comprising a 113th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 225-226, a 114th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 227-228, a 115th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 229-230, and a 116th primer pair having a nucleotide sequence as shown in SEQ ID NOs: 231-232; Zaru virus primers, the Zaru virus primer set includes the 117th primer pair with nucleotide sequences as shown in SEQ ID NOs: 233-234, the 118th primer pair with nucleotide sequences as shown in SEQ ID NOs: 235-236, the 119th primer pair with nucleotide sequences as shown in SEQ ID NOs: 237-238, and the 200th primer pair with nucleotide sequences as shown in SEQ ID NOs: 239-240; A human astrovirus primer set, comprising a 201st primer pair having a nucleotide sequence as shown in SEQ ID NOs: 241-242.

2. The primer composition for detecting infectious diarrhea-type pathogens according to claim 1, characterized in that: Each of the first primer pair to the 201st primer pair consists of a forward primer and a corresponding reverse primer.

3. The primer composition for detecting infectious diarrhea-type pathogens according to claim 1, characterized in that: Each primer in each primer pair from the first primer pair to the 201st primer pair adds a Barocde sequence, and the Barocde sequences of different infectious diarrhea-type pathogens are different.

4. The primer composition for detecting infectious diarrhea-type pathogens according to claim 1, characterized in that: The 5' end of each primer in each primer pair from the first primer pair to the 201st primer pair is phosphorylated.

5. A kit for detecting infectious diarrhea-type pathogens, characterized in that: The method comprises the primer composition for detecting infectious diarrhea-type pathogens according to any one of claims 1 to 4, an artificial plasmid set and a multiplex polymerase chain reaction mixture.

6. The kit for detecting infectious diarrhea-type pathogens according to claim 5, characterized in that: The artificial plasmid set includes artificial plasmids of different infectious diarrhea pathogens, and the artificial plasmids have the same primer ends as the corresponding infectious diarrhea pathogen amplicons, the middle regions have 18-22% differences, and the GC content differences are 1%-3%.

7. The kit for detecting infectious diarrhea-type pathogens according to claim 5, characterized in that: The artificial plasmid set includes one or any combination of artificial plasmids of Staphylococcus aureus, artificial plasmids of Escherichia coli, artificial plasmids of Klebsiella pneumoniae, artificial plasmids of Proteus mirabilis, artificial plasmids of Listeria monocytogenes, artificial plasmids of Pseudomonas aeruginosa, artificial plasmids of Salmonella enterica, artificial plasmids of Bacillus perfringens, artificial plasmids of Vibrio cholerae, and artificial plasmids of Clostridium difficile.

8. The kit for detecting infectious diarrhea-type pathogens according to claim 5, characterized in that: The nucleotide sequence of the artificial plasmid of Staphylococcus aureus is shown in SEQ ID NO.243, the nucleotide sequence of the artificial plasmid of Escherichia coli is shown in SEQ ID NO.244, the nucleotide sequence of the artificial plasmid of Klebsiella pneumoniae is shown in SEQ ID NO.245, the nucleotide sequence of the artificial plasmid of Proteus mirabilis is shown in SEQ ID NO.246, the nucleotide sequence of the artificial plasmid of Listeria monocytogenes is shown in SEQ ID NO.247, the nucleotide sequence of the artificial plasmid of Pseudomonas aeruginosa is shown in SEQ ID NO.248, the nucleotide sequence of the artificial plasmid of Salmonella enterica is shown in SEQ ID NO.249, the nucleotide sequence of the artificial plasmid of Bacillus perfringens is shown in SEQ ID NO.250, the nucleotide sequence of the artificial plasmid of Vibrio cholerae is shown in SEQ ID NO.251, and the nucleotide sequence of the artificial plasmid of Clostridium difficile is shown in SEQ ID NO.

252.

9. The kit for detecting infectious diarrhea-type pathogens according to claim 5, characterized in that: The primer composition for detecting infectious diarrhea-type pathogens, the artificial plasmid set, the multiple polymerase chain reaction mixture, the DNA template and the sample to be tested are mixed to perform multiple PCR amplification to obtain PCR products.

10. The kit for detecting infectious diarrhea-type pathogens according to claim 9, characterized in that: The PCR product is directly added to the sequencing adapter for sequencing, wherein the sequencing is performed using a nanopore sequencing platform.

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