Multiple PCR primer screening method and application

Abstract

The invention relates to a multiple PCR primer screening method and application. The method comprises the steps that S1, a single PCR reaction is conducted on a single target fragment; S2, a pair of primers is selected for each target fragment, the primers are randomly combined, and 3-4 pairs of primers in each group are subjected to small multiple PCR system optimization; and S3, the primers obtained through screening in the step S2 are mixed, total multiple PCR is conducted, and the primers which are high in amplification efficiency and free of specific amplification are selected. Meanwhile,the invention further provides a multiple PCR detection kit for 13 respiratory tract pathogenic microorganisms obtained through the screening method. The kit comprises 13 pairs of primers which are specifically shown in SEQ ID NO. 1-26. According to the screening method, the purpose of rapidly detecting and screening the primers can be achieved, the optimization period of a multiple PCR system issignificantly shortened, and the optimization difficulty is lowered; and the detection kit can detect the 13 common respiratory tract pathogenic microorganisms at the same time, the detection cost islowered, the detection time is saved, the detection efficiency is high, and the method is suitable for clinical large-scale application.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a screening method and application of multiple PCR primers. Background technique [0002] The sources of respiratory infections are extremely complex, including viruses, bacteria, mycoplasma, chlamydia and other microorganisms. The currently known pathogenic microorganisms related to respiratory infections include: Streptococcus pneumoniae, parainfluenza virus, influenza A virus, influenza B virus, respiratory syndrome Cytovirus, Haemophilus influenzae, Mycoplasma pneumoniae and adenovirus etc. [0003] Among them, Streptococcus pneumoniae can cause pneumonia, meningitis, otitis media, sinusitis and other serious diseases, among which LytA (N-acetylmuramoyl-L-alanine amidase) is closely related to bacterial virulence It is the first bacterial autolytic enzyme qualitative at the molecular level. It exists in all serotypes of Streptococcus pneumoniae and is located in the c...

AI Technical Summary

Problems solved by technology

However, the disadvantage of ordinary PCR is that one reaction can only detect a single gene, while multiplex PCR technology can detect multiple genes in one reaction at the same time to quickly and efficiently detect pathogenic microorganisms

However, there are some problems in the establishment of the multiplex PCR system: (1) The design of primers is difficult: at the same reaction temperature, it is necessary to take into account that all primers maintain high amplification efficiency, reduce non-specific amplification and primers are mutually incompatible; interference; (2) Low optimization efficiency: perform single-plex PCR reaction first, then increase primer pairs sequentially, constantly adjust reaction conditions or replace primers; (3) Long PCR reaction time: traditional PCR reaction is a three-step method, and multiple PCR reactions Because there are multiple fragments to be amplified, the lack of enzymes and dNTPs becomes a limiting factor, so the annealing and extension time needs to be extended to complete the synthesis of all products, which further lengthens the reaction time

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