A kit for simultaneous detection of eighteen kinds of pathogens with fever and rash and its detection method

Abstract

The invention discloses a kit capable of synchronously detecting eighteen kinds of fever with eruption pathogens and a detection method thereof. The kit comprises diethylpyrocarbonate (DEPC) water, 5*reverse transcriptase (RT) buffer solution, reverse transcription primers, reverse transcriptase, X solution, 10* polymerase chain reaction (PCR) buffer solution, PCR primers, 25m M magnesium chloride solution, deoxyribonucleic acid (DNA) polymerase and positive reference substances. The kit is characterized in that the reverse transcription primers comprise five kinds of fever with eruption pathogens and ribonucleic acid (RNA) internal reference RT primers, and the sequences are shown as SEQ ID NO.1-NO.6. The PCR primers comprises the remaining thirteen kinds of fever with eruption pathogens, herpes simplex virus 1, 2 universal type, human DNA internal reference, forward and reverse PCR amplification primers of a reaction internal reference, and the five kinds of fever with eruption pathogens and PCR amplification primers of a human RNA internal reference. The gene sequence is shown as SEQ ID NO.7-NO.44. The kit has the advantages of being strong in specificity, high in sensitivity, high in flux, strong in reliability, low in cost, and free from false-negative results.

Description

technical field [0001] The invention relates to a detection kit and a detection method for pathogens with fever and rash, in particular to a kit for synchronously detecting eighteen kinds of pathogens with fever and rash based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Fever with rash is a disease with fever and symptoms as the main clinical manifestations, including measles, rubella and other RFIs, and often occurs in the form of outbreaks. The existing fever with rash disease has a high incidence rate, especially for infants and young children, and poses a huge threat to human health and social development. The clinical symptoms of such diseases are generally mild, but severe complications and their histological similarities make accurate diagnosis of pathogens and dialectical treatment critical. At present, most methods of diagnosis of fever with rash, such as pathogen culture, serological diag...

AI Technical Summary

Problems solved by technology

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

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 18 kinds of pathogens with fever and rash based on the GeXP multiple gene expression genetic analysis system at home and abroad

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