Sputum liquefaction method and special liquefaction reagent thereof

Abstract

The invention discloses a sputum liquefaction method and a special liquefaction reagent thereof, and belongs to the field of biotechnology methods. The sputum liquefying method comprises the following steps: uniformly mixing a sputum liquefying reagent and a sputum sample, and then carrying out high-temperature treatment or high-temperature and ultrasonic combined treatment, so that the aims of completely liquefying sputum, releasing nucleic acid from pathogens and simultaneously protecting the nucleic acid of the pathogens from being degraded in a long-acting manner can be fulfilled through one-step operation, the operation is simple and convenient, the consumed time is short, and the cost is low. The liquefaction efficiency of the sputum sample can be greatly improved, and the positive detection rate can be remarkably improved. The sputum liquefying reagent is simple in composition and low in component concentration, and the production cost can be greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology methods, and in particular relates to a sputum liquefaction method and a special liquefaction reagent thereof. Background technique [0002] Nucleic acid detection is one of the important methods for pathogen detection. At present, there are two types of pathogen nucleic acid detection, one is DNA as the detection target, and the other is RNA as the detection target. The copy number of DNA in pathogens is generally low, while the copy number of RNA in pathogens is high, especially ribosomal RNA. A pathogen can have hundreds, thousands, or even tens of thousands of RNA target copies, so the RNA of a pathogen Targeted pathogen detection methods can improve the detection rate of pathogens. However, due to the extremely easy degradation of RNA, once the pathogen dies, the RNA will be degraded quickly, so that the pathogen cannot be detected. [0003] The detection of pathogens in sputum is an important ...

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Problems solved by technology

However, when this method is used for liquefaction of sputum samples, most of the pathogens in the sputum will die quickly, leading to RNA degradation, which will eventually lead to a low detection rate of pathogen RNA, and the liquefaction samples obtained by this liquefaction method are difficult to be used directly. Subsequent pathogen nucleic acid detection often requires enrichment of pathogen precipitation after liquefaction, and then nucleic acid extraction. The operation is more complicated, and many pathogen sputum samples with low tolerance to sodium hydroxide solution are not suitable for this liquefaction method.

The principle of protease method is to use protease to digest sputum mucin. The cost of protease required by this method is high, and the enzymatic hydrolysis reaction takes a long time (generally 1-5 hours at 37-65°C), and it is easy to liquefy unevenly, and the efficiency is low. Low

The DTT method uses DTT containing thiol (-SH) to break the mucin, the main component of the sputum, which causes viscosity. This method requires the use of high-concentration DTT, which takes a long time and often requires centrifugation. Stored under low temperature conditions, the sputum liquefied by this method is often unevenly liquefied, and it is easy to change from a solution state to a gel state, and DTT has certain toxicity, so it is not suitable for high-concentration use in clinical liquefaction of sputum samples

The combined method of protease method and DTT method can reduce the transition of sputum from solution state to gel state after liquefaction to a certain extent, but this method still needs to use protease with high cost and high concentration of DTT, and the composition is complex. Since the use of protease to liquefy sputum requires long-term incubation at room temperature or above, and the poor stability of DTT requires low-temperature storage, the protease liquefaction operation and DTT liquefaction operation must be carried out step by step, which also makes the method cumbersome. , takes a long time, the liquefaction efficiency is low, and operations such as centrifugation may be required, so it is not suitable for clinical liquefaction of sputum samples directly for nucleic acid detection

Although sputum liquefaction reagents composed of guanidines and cysteine ​​derivatives can protect nucleic acids in samples for a long time at room temperature, they are not suitable for effective protection of nucleic acids released from lysed pathogens under high temperature treatment conditions , resulting in a low positive detection rate

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