Sputum pretreatment method for quickly carrying out nucleic acid detection

Abstract

The invention discloses a sputum pretreatment method for quickly carrying out nucleic acid detection. The sputum pretreatment method comprises the following steps of a first step, adding a NaOH solution with a concentration of 4 to 30 percent into a collected sputum specimen, wherein the amount of the NaOH solution is 4 times that of the collected sputum specimen, carrying out vortex oscillation to liquefy the collected sputum specimen, and standing for 5min to 10min at a room temperature; a second step, adding a citric acid solution with a concentration of 20 to 80 percent into specimen liquid treated in the first step, and uniformly oscillating and mixing; a third step, adding thiourea with a concentration of 5 to 14 percent into specimen liquid treated in the second step, uniformly oscillating and mixing, then standing at the room temperature, and removing a deposit. The sputum pretreatment method has the advantages that the method is simple and quick, as the entire pretreatment process does not need centrifugation, the operation is convenient, and the consumed time is shorter (less than 15 minutes); the specimen liquid pretreated by the method provided by the invention is higher in sensitivity; 30 bacteria per milliliter can be lowest detected; the specificity is good, and according to a clinical specimen verification result, the negative coincidence rate can reach 100 percent.

Description

technical field [0001] The invention relates to the processing of sputum samples, in particular to a method for pre-processing sputum for rapid nucleic acid detection. Background technique [0002] Tuberculosis caused by Mycobacterium tuberculosis is one of the common human infectious diseases. About one-third of the world's population has been infected, and Asians account for two-thirds of the world's infections. Therefore, tuberculosis has become a global threat to human health. public health issues. Clinical detection of Mycobacterium tuberculosis mainly includes microbiological culture, immunological detection and molecular biology, which has developed rapidly in recent years, nucleic acid detection technology. [0003] At present, the main sample type for clinical detection of Mycobacterium tuberculosis is sputum specimen, but the composition of sputum is relatively complex, which mainly includes mucus, foreign bodies, pathogenic microorganisms, various inflammatory ce...

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Problems solved by technology

[0003] At present, the main sample type for clinical detection of Mycobacterium tuberculosis is sputum specimen, but the composition of sputum is relatively complex, which mainly includes mucus, foreign bodies, pathogenic microorganisms, various inflammatory cells, and necrotic and exfoliated mucosal epithelial cells. mucin, acid glycoprotein, Ca 2＋ The high content of sputum leads to high viscosity of sputum, so sputum needs special pretreatment before participating in nucleic acid extraction, so that it can participate in subsequent PCR amplification detection

The traditional sputum treatment method is alkaline lysis, which is mainly NaOH liquefaction, high-speed centrifugation to collect the precipitate, normal saline or phosphate solution to wash the precipitate, and then centrifuge to collect the bacteria. This method has complicated processing steps, inconvenient operation and time-consuming Long (≥60min), high bacterial cell loss rate and low impurity removal rate during repeated centrifugation and washing, resulting in false negatives in subsequent test results

At present, the nucleic acid extraction method used in the nucleic acid detection kits on the market is the boiling method, which cannot be matched with automatic nucleic acid detection technology and has low sensitivity, which may easily cause false negative results

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