Quantitative real-time fluorescence PCR (Polymerase Chain Reaction) quick detection kit of Weissella viridescens, and detection method thereof

Abstract

The invention provides a quantitative real-time fluorescence PCR (Polymerase Chain Reaction) quick detection kit of Weissella viridescens, and a detection method thereof. The quantitative real-time fluorescence PCR quick detection kit of the Weissella viridescens comprises reaction liquid 1 and reaction liquid 2, wherein the reaction liquid 1 comprises 10mmol / L of deoxyribonucleoside triphosphate,2.5mu L of 10*Taq Buffer reaction buffer solution, 1mu L of 20*SYBR Green I fluorochrome and 15.5 mu L of RNase-Free ddH2O; the reaction liquid 2 comprises 1.5mu L of 10mu mol / L upstream primer, 1.5mu L of 10 mu mol / L downstream primer and 5U / mu L Taq DNA (deoxyribonucleic acid) polymerase. The invention also specifically discloses a quantitative detection method and a qualitative detection method for the quantitative real-time fluorescence PCR quick detection kit of Weissella viridescens. Compared with a conventional PCR technology, the method has the advantages of high specificity, high sensitivity, simpleness in operation, high speed in detection, and accuracy in quantification, can be used for the quick detection of the Weissella viridescens, and has a high practical value.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a biological detection reagent for rapid detection of bacterial samples by real-time fluorescent quantitative PCR (Quantitative Real-time PCR) technology, in particular to a real-time fluorescent quantitative PCR rapid detection kit for Weissella viride and its detection method. Background technique [0002] Weissella viridescens belongs to the Lactobacillales (Lactobacillales) Leuconostocaceae (Leuconostocaceae) Weissella genus (Weissella), which is similar to other representative strains of lactic acid bacteria in terms of microscopic and macroscopic morphology, especially Easily confused with Leuconostoc and Lactobacillus. Weissella viride is a Gram-positive bacterium, which is irregularly short rod-shaped, with round or slightly smaller ends, arranged in pairs or short chains, and forms small transparent colonies on MRS agar medium, showing microscopic Raised round ...

AI Technical Summary

Problems solved by technology

The traditional identification method needs to go through pre-enrichment, re-enrichment of selective medium, streak culture of the culture on a plate containing one or more agents that inhibit the growth of the tested bacteria, confirmation of characteristic colonies visible to the naked eye, And carry out a series of biochemical and serotype tests on the colony to make identification, etc., the operation is cumbersome, and the reliability of the results is low, and it takes a long time, which seriously affects the cycle of detection and identification; polymerase chain reaction (PCR) is a Molecular biology technology for amplifying and amplifying specific DNA fragments. A high copy number is amplified by PCR reaction, and then the target fragment is detected by agarose gel electrophoresis.

However, the detection specificity and sensitivity of the electrophoresis method are not high, so non-specific hybrids such as primer dimers are likely to cause misjudgment, and the experimental process cannot achieve complete closed-tube operation, which increases the risk of PCR product contamination

DNA polymerase will be inactivated at high temperature. Therefore, new DNA polymerase must be added in each cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology.

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