Kit for synchronously detecting thirty diarrhea pathogens and detection method of kit

Abstract

The invention discloses a kit for synchronously detecting thirty diarrhea pathogens and a detection method of the kit. The kit comprises DEPC (diethylpyrocarbonate) water, a 5*RT (reverse transcription) buffer, a reverse transcription primer, a reverse transcriptase, an X solution, a 10*PCR (polymerase chain reaction) buffer, a PCR primer, a 25mM magnesium chloride solution, a DNA (deoxyribonucleic acid) polymerase and a positive control, and is characterized in that the reverse transcription primer comprises RT amplification primers of eleven diarrhea RNA viruses and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-12 (sequence identifier number 1-12), and the PCR primer comprises forward and reverse PCR amplification primers of the rest nineteen diarrhea pathogens, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of eleven diarrhea RNA viruses and the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 13-66. The kit and the detection method have the advantages of high specificity, sensitivity, flux and reliability, low cost, and no false negative results.

Description

technical field [0001] The invention relates to a detection kit for diarrhea pathogenic bacteria and a detection method thereof, in particular to a kit for synchronously detecting thirty kinds of diarrhea pathogenic bacteria based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Diarrhea is a gastrointestinal infectious disease with a high incidence and is prevalent all over the world. Its incidence is second only to upper respiratory tract infection. In my country, the incidence of infectious diarrhea ranks first among all infectious diseases. Infectious diarrhea is a common and frequently-occurring disease in my country. The etiology is relatively complex and can be caused by viruses, bacteria and protozoa. A rapid and efficient diagnostic method for diarrhea-causing bacteria. [0003] At present, the traditional detection methods for pathogenic bacteria in diarrhea mainly include the following: [00...

AI Technical Summary

Problems solved by technology

During light microscope examination, mucus stool, bloody stool or bloody stool may have a lot of red and white blood cells, which are more common in bacteria such as Salmonella, invasive Escherichia coli, enterohaemorrhagic Escherichia coli, Campylobacter, Yersinia and some viruses Diarrhea caused by diarrhea, etc.; loose stools, watery stools, may have a small amount or no red and white blood cells, and are more common in diarrhea caused by enterotoxigenic Escherichia coli, rotavirus, Cryptosporidium, Aeromonas, etc., with cost Low, low requirements for laboratory hardware, but there are the following disadvantages: 1) low sensitivity, often false negative; 2) low accuracy, easy to cause misdiagnosis or misdiagnosis

Disadvantages: 1) Long time-consuming: generally 3-5 days or longer; 2) Low diagnostic efficiency: only a single bacterium can be purely cultured; for some pathogens with variable phenotypic characteristics, it is difficult to distinguish with morphological experience; in addition, due to Abuse of antibiotics, the growth of bacteria is inhibited during the detection process, resulting in false negative results; 3) Huge workload: Since every bacteria in each sampling sample must be tested, the workload is very huge, especially for some cultures. Bacteria with strict environmental requirements will increase the workload and difficulty of detection

Advantages: 1) High sample throughput: using 96-well microplate, one plate can complete the detection of multiple samples; 2) More sensitive, using the characteristics of enzyme linkage, the original antigen signal can be amplified; 3) Fast: In terms of time, it is much shorter than the traditional medium microbial culture inspection method; but it also has the following disadvantages: 1) False positives are prone to occur: due to washing and antigen coating operations, or cross-reaction due to similar antigenic surface determinants, Therefore, false positives are prone to occur; 2) Time-consuming and labor-intensive: making antibodies is a very time-consuming and labor-intensive work; 3) Uncertain sensitivity: the sensitivity of the experiment depends on the quality of the antibody, and each antibody is different. Difficult to control quality; 4) Unable to detect multi-variant viruses: Viruses that mutate rapidly, such as some RNA viruses, have multiple subtypes and often mutate. Mutations cause antibodies to fail and antigens cannot be detected

However, there are also the following disadvantages: 1) Low throughput: only one pathogenic component can be detected at a time. If multiple pathogenic bacteria need to be detected, multiple PCR instruments are required to work at the same time, with low efficiency and long cycle, which will pollute large quantities of multiple pathogens. 2) The cost is relatively high: because only one pathogen can be detected at a time, when a sample needs to detect multiple pathogens at the same time, it must be tested one by one, and the cost is relatively high

Advantages: 1) High-throughput parallel detection: When a sample needs to detect multiple pathogenic bacteria at the same time, all the results can be obtained in one experiment; 2) Simple and fast operation: the whole detection can basically produce results in 4-8 hours; but There are also the following disadvantages: 1) The technology is expensive and complicated: each sample requires a chip, and the cost is more than ￥1000 / sample, which is not conducive to large-scale promotion; 2) The synthesis and immobilization of probes are more complicated, especially the production of high-density Probe array is the main rate-limiting step; 3) It cannot be accurately quantified and the repeatability is poor; 4) The sensitivity is low: the chip method requires a large amount of nucleic acid, and generally requires multiple PCR amplification first. Dimers, hairpin structures, or different Tm values ​​lead to different amplified target fragment efficiencies, which in turn affect detection sensitivity; 5) Due to the variety of chips, it is difficult to formulate a unified quality control standard

[0016] At present, there are no relevant research reports on the simultaneous detection of thirty kinds of diarrhea pathogenic bacteria based on the GeXP multiple gene expression genetic analysis system and its detection methods at home and abroad

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