Preparation method and application of PCR positive control substance capable of distinguishing pollution

Abstract

The invention discloses a preparation method of a PCR positive control substance; the pollution of the positive control substance to a nucleic acid amplification system can be obviously distinguished,the accuracy of PCR detection is improved and the false positive is reduced. The nucleic acid sequence of a PCR positive fragment has a section of sequence removed from two primers, and then clone engineering bacteria are transformed. A positive plasmid is extracted as a control. The transformed control does not affect the combination of PCR primers, also does not affect the amplification efficiency of PCR products. Moreover, because the length of the fragment is smaller than that of normal positive sample products, the fragment can be distinguished significantly when agarose electrophoresisis carried out, and thus a role in distinguishing pollution is played.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically provides a method for preparing a positive control substance of a nucleic acid amplification system. The control substance prepared by the method and a normal sample are subjected to PCR amplification at the same time, and target bands can be effectively distinguished. Background technique [0002] PCR is an extremely sensitive nucleic acid amplification technique that is susceptible to contamination. Positive control substance is one of the main sources of contamination. Due to the large number of copies, aerosols are formed in contact with the air during the process of opening, shaking, and sampling during PCR amplification, and walk in the environment of the experimental area, causing serious pollution, resulting in false positive results, and seriously interfering with PCR. detection accuracy. In order to solve this problem, researchers at home and abroad have proposed many kin...

AI Technical Summary

Problems solved by technology

Although these technologies have been proven to be effective in controlling pollution, these methods only play a preventive role, and it is impossible to accurately distinguish pollution that has already occurred.

[0003] Positive control substances mostly use purified and specified concentrations of positive plasmids, nucleic acid amplification products or artificially synthesized nucleic acid fragments, and the concentration is as high as 10 8 copy / ul, so it is difficult to avoid the contamination of normal samples by positive control substances during the development and use of diagnostic reagents

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