Method for quickly detecting klebsiella pneumoniae triple qPCR in respiratory tract sample

Abstract

The invention provides a method for quickly detecting klebsiella pneumoniae triple qPCR in a respiratory tract sample. The method comprises the following steps: collecting and selecting a clinical respiratory tract sample; performing bacterial culture and identifying; extracting and purifying nucleic acid; constructing quality control/interior label plasmid; designing and compounding a sequencing primer; optimizing a clinical isolate high-fidelity system, amplifying and judging a result; sequencing a high-fidelity PCR product and controlling quality; designing a triple qPCR primer and a probe and compounding a marker; and optimizing a triple qPCR system, amplifying and judging the result. The method has the beneficial effects that klebsiella pneumoniae rcsA and 23s RNA housekeeping genes can be simultaneously detected, and the adopted primer and probe are designed in the manner of effectively avoiding a variation area or a mutational site according to a gene sequencing result of popular distribution strain in China; a quick sputum liquefying bactericide is added into a sputum sample, so that the sputum can be quickly liquefied, pathogenic microorganisms can be killed, and an operator can be protected from being infected; and compared with the traditional method, the method provided by the invention can reduce the time by about 1 hour and has the characteristics of simple and convenient operation, high speed, safety and sensitivity.

Description

[0001] 【Technical field】 [0002] The invention relates to a triple qPCR method for Klebsiella pneumoniae, in particular to a triple qPCR method for rapid detection of Klebsiella pneumoniae in respiratory samples. [0003] 【Background technique】 [0004] Klebsiella pneumoniae (Klebsiella pneumoniae) is one of the pathogenic bacteria of respiratory tract infection, which can cause severe lower respiratory tract infection and even death. In recent years, due to the lack of early and accurate treatment, the problem of drug resistance has become increasingly prominent, seriously affecting the treatment effect [1-2] . The culture method is a commonly used detection method at present, and it is also the gold standard. It usually takes 2-3 days or even longer, and its timeliness, simplicity, and sensitivity cannot meet the needs of clinical rapid and accurate diagnosis. [3] . Real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) detects genetic material (DNA and...

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to solve the existing triple qPCR method and reagents for Klebsiella pneumoniae in respiratory samples, which only detects a single gene, and does not sequence and compare the Klebsiella pneumoniae-related genes that are popular in my country, only according to NCBI or literature data to design primers and probes, due to biological evolution, regional distribution, etc., there may be fatal shortcomings of insufficient sensitivity or specificity, and a high proportion of false negatives and false positives will bring misdiagnosis and / or misdiagnosis to clinical practice. A newly established triple qPCR method for rapid detection of Klebsiella pneumoniae in respiratory samples due to the lack of serious problems such as mistreatment or mistreatment

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