A detection primer set, detection kit and multiple PCR detection method for Streptococcus agalactiae

Abstract

The invention discloses a detection primer set for Streptococcus agalactiae, comprising a primer pair hylB, a primer pair ponA and a primer pair cfb; wherein, the primer pair hylB includes a primer whose nucleotide sequence is shown in SEQ ID NO.1 hylB-F and nucleotide sequence such as primer hylB-R shown in SEQ ID NO.2; said primer pair ponA includes nucleotide sequence such as primer ponA-F and nucleotide sequence shown in SEQ ID NO.3 Primer ponA-R as shown in SEQ ID NO.4; Described primer pair cfb comprises nucleotide sequence as shown in the primer cfb-F of SEQ ID NO.5 and nucleotide sequence as shown in SEQ ID NO.6 Primer cfb‑R. The invention also discloses a detection kit comprising the primer set and a multiplex PCR detection method using the detection kit. The invention has good specificity, high sensitivity, simplicity, speed, efficiency and precision, and is suitable for dairy-free products in pollution-free aquatic products. The rapid inspection and quarantine of streptococcus can be directly applied to the early monitoring and early warning of aquaculture diseases. The minimum concentration for detection of Streptococcus agalactiae DNA is 7.74×10 ‑3 ng / uL, no need for bacterial culture, less sample required for detection, and minimally invasive sampling can be achieved.

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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