Detection primer set of Streptococcus agalactiae, detection kit and multiplex PCR detection method

Abstract

The invention discloses a detection primer set of Streptococcus agalactiae. The detection primer set of Streptococcus agalactiae comprises a primer pair hylB, a primer pair ponA and a primer pair cfb, wherein the primer pair hylB comprises a primer hylB-F of which the nucleotide sequence is as shown in SEQ ID NO.1 and a primer hylB-R of which the nucleotide sequence is as shown in SEQ ID NO.2; the primer pair ponA comprises a primer ponA-F of which the nucleotide sequence is as shown in SEQ ID NO.3 and a primer ponA-R of which the nucleotide sequence is as shown in SEQ ID NO.4; the primer pair cfb comprises a primer cfb-F of which the nucleotide sequence is shown in SEQ ID NO.5 and a primer cfb-R of which the nucleotide sequence is shown in SEQ ID NO.6. The invention further discloses a detection kit containing the primer set and a multiplex PCR detection method using the detection kit. The primer set and the detection method disclosed by the invention are good in specificity, high in sensitivity, simple and fast, high-efficient and accurate, suitable for rapid inspection and quarantine of Streptococcus agalactiae in pollution-free aquatic products, and can be directly applied to early monitoring and early warning of aquaculture diseases. The lowest concentration of Streptococcus agalactiae DNA detected by the detection kit is 7.74*10<-3>ng / uL, and the detection kit can achieve minimally invasive sampling without bacterial culture, and requiring small quantity of samples for detection.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria in aquaculture animals, and in particular relates to a detection primer set for Streptococcus agalactiae. At the same time, the present invention also relates to a detection kit comprising the above primer set and a multiplex PCR detection method using the kit. Background technique [0002] Streptococcus agalactiae ( Streptococcus agalactiae ) belongs to Bacillus, Lactobacillus, Streptococcus, and Streptococcus. It is an important zoonotic pathogen. So far, it has been found that more than 30 kinds of farmed freshwater fish and more than 50 kinds of seawater fish are susceptible to the infection of Streptococcus agalactiae, and the annual loss caused by Streptococcus agalactiae in the world's aquaculture industry is as high as 10 billion U.S. dollars. Non-fish are most susceptible to the invasion of the pathogen. The onset of Streptococcus agalactiae is relatively fast, and ther...

AI Technical Summary

Problems solved by technology

Although the molecular detection methods of Streptococcus agalactiae have been reported at home and abroad, most of them use purely cultured bacteria liquid for detection. Using ordinary PCR method, only one gene can be detected at a time, which often leads to missed detection or false detection.

[0003] Multiplex PCR is a target sequence that can simultaneously amplify multiple target fragments in one reaction. This technology can be used for the detection or identification of various pathogenic microorganisms. Multiple pairs of specific primers are added to the same PCR reaction tube at the same time for PCR amplification. , the biggest problem lies in the design of primers and the optimization of reaction conditions, because the band loss phenomenon will occur after the amplification of various primers is mixed, and there is still a competitive relationship between the primers, and the strong primers may cover the weak primers, resulting in the loss of the target fragment. Even primer-primer interactions can generate severe primer-dimers

At present, there are reports in the literature on the construction of a triple PCR detection method for Streptococcus agalactiae, but the minimum detection concentration of this detection technique for DNA samples of Streptococcus agalactiae is only 3.2×10 -1 ng / μL

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