Kit for synchronously detecting twenty-two respiratory tract pathogens and detection method of kit

Abstract

The invention discloses a kit for synchronously detecting twenty-two respiratory tract 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 the nine respiratory tract pathogens, and a reverse primer of a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-6 (sequence identifier number 1-6), and the PCR primer comprises forward and reverse PCR amplification primers of the rest thirteen respiratory tract pathogens, a forward and reverse amplification primer of a human DNA internal reference, a forward and reverse PCR amplification primer of a reaction internal reference, PCR amplification primers of the nine respiratory tract pathogens, and a PCR amplification primer of the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 7-44. 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 respiratory pathogens and a detection method thereof, in particular to a kit for synchronous detection of 22 kinds of respiratory pathogens based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Respiratory tract infections cause a large number of people to get sick or die every year, especially when there are local outbreaks or explosive epidemics of influenza A virus, which poses a huge threat to people's health and society; other pathogen infections can also cause outbreaks. At present, the final diagnosis method for respiratory infections is still based on pathogen inspection, and the epidemic has already spread to a certain extent when the diagnosis is reported. Therefore, it is urgent to establish rapid and efficient diagnostic methods for respiratory pathogens. [0003] At present, the traditional detection methods for respirator...

AI Technical Summary

Problems solved by technology

But there are the following disadvantages: 1) Long time-consuming: generally 3-5 days or longer

2) Low diagnostic efficiency: each bacterium can only be cultured separately; it is difficult to distinguish some pathogens with phenotypic characteristics with morphological experience; in addition, due to the abuse of antibiotics, the growth of bacteria is inhibited during the detection process, resulting in false negatives 3) Huge workload: Since each type of bacteria in each sampling sample must be tested, the workload is very huge, especially for some bacteria that have strict requirements on the culture environment, which increases the workload and difficulty of detection.

However, there are the following disadvantages: 1) False positives are prone to occur: due to the operation of washing and antigen coating, or cross-reaction due to similar antigenic surface determinants, false positives are prone to occur; 2) Time-consuming and labor-intensive: making antibodies is very expensive Time-consuming and labor-intensive work; 3) Uncertain sensitivity: the sensitivity of the experiment depends on the quality of the antibody, and the quality of each antibody is different, and the quality is difficult to control; 4) Unable to detect multi-variant viruses: the variability is faster Some viruses, such as influenza A virus, have multiple subtypes and often mutate, which leads to the failure of antibodies and the inability to detect antigens

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

[0015] At present, there are no relevant research reports on the kits and detection methods for the simultaneous detection of 22 respiratory pathogens based on the GeXP multiple gene expression genetic analysis system at home and abroad

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