Multiplex fluorescent quantitative probe method PCR (Polymerase Chain Reaction) kit for detecting urethral pathogen infection

Abstract

The invention discloses a urogenital tract infection pathogen multiple detection kit and application thereof. Specifically, the invention develops a fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting 12 clinical common urethral infection germs. The optimized primer and probe provided by the invention have more excellent combination of 12 groups of PCR primers and probes, have excellent stability, and do not interfere with each other in a PCR reaction process, so that the optimized primer and probe can be used for a multiplex fluorescent quantitative PCR method.

Description

technical field [0001] The invention relates to the field of biological detection. Specifically, the present invention relates to a multiplex fluorescent quantitative (probe method) PCR kit for detecting urinary tract pathogen infection and its application. Background technique [0002] Urinary tract infection (UTI), referred to as urinary infection—also known as urinary tract infection, refers to the inflammation of the urinary tract caused by pathogens invading the urinary tract mucosa or tissue. It is a common disease, and the prevalence rate in the general population is as high as 1%. . A variety of pathogens such as bacteria, fungi, mycoplasma, chlamydia, viruses, parasites, etc. can cause urinary sensation, usually accompanied by bacteriuria and pyuria. According to the site, it can be divided into upper urinary tract infection (mainly pyelonephritis, Pyelonephritis) and lower urinary tract infection (mainly cystitis, Cystitis). According to whether there is urinary...

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Problems solved by technology

However, these methods have certain defects and limitations: microscopic examination and culture are the basic methods for routine fungal examination, but the positive rate is low and time-consuming; histopathological examination is an invasive operation, which is not suitable for obtaining materials, and the patients are more painful. Moreover, immunohistochemical methods are needed for further verification; although imaging examinations are more convenient, they lack specificity; although serological examinations are quick and easy, their sensitivity, specificity and false positives still have a lot to do with early diagnosis and disease monitoring. Deficiencies, and it is impossible to identify the type of infectious pathogen

[0005] Multiplex PCR (multiplex PCR), also known as multiple primer PCR or composite PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. It can be used for its reaction principle, reaction The reagents and operation process are similar to general PCR, but the multiplex PCR reaction is affected by many factors, which can easily interfere with the results, such as unbalanced reaction system, uneven product amplification, and difficult detection; poor primer specificity, easy to mix with other The combination of non-target gene fragments; the optimal annealing temperature between multiple sets of primers needs to be close to each other; the hairpin structure formed between the primers interferes with the competition between the primers and the target binding site, affecting the amplification efficiency

[0006] There are the following problems in the prior art of primer sets used for multiple urinary tract infection single system detection of multiple pathogens: (1) more multiple reactions cannot be realized, such as one tube can only detect two target pathogens and one internal standard, and only supports triple (CN111593144A); (2) The coverage of target pathogens is relatively narrow, such as only supporting the detection of mycoplasma (CN110343777A), and only supporting the detection of reproductive tract-related pathogens (CN111647672A); The steps and instruments are relatively complicated (CN202110167340)

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