Detection primer set and kit for salmonella typhimurium MLVA typing, and applications of detection primer set
A Salmonella typhimurium detection primer technology, applied in the field of microbiology, can solve the problems that affect the accuracy of sequence analysis, affect the reliability of results, and cannot improve the resolution of the method.
Active Publication Date: 2019-04-12
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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Problems solved by technology
In the experimental verification of a large number of samples, it was found that the STTR9, STTR5, STTR6, and STTR10 loci in the original MLVA-5 locus typing method had high resolution and short repeat sequences (6-9bp), but the STTR3 locus repeat sequence But it is very long, and there are 2 types, namely 27bp and 33bp. The repeated fragments of long sequences affect the accuracy of sequence analysis, thus affecting the reliability of the results, and the polymorphism of the STTR3 site is found in the study. Can not improve Resolution of the whole method
At the same time, the amplification process of each VNTR site in the MLVA-5 site method cannot be completed in the same reaction system
Method used
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Embodiment 1
[0038] 1. Design of specific primer sequences:
[0039] The sequence information of the known standard strain LT2 of Salmonella typhimurium was analyzed, and specific primer sequences were designed for four VNTR loci with good resolution ability—MLVA1, MLVA2, MLVA3, and MLVA4. Primerpremier5.0 software was used to design primers in the conserved regions on both sides of the four VNTR regions, and try to make the length of the target fragments have a large difference to facilitate the judgment of the results. Ensure that each primer sequence has a similar Tm value, the primer itself avoids the formation of a secondary structure hairpin structure, and there is no cross-reaction between primers in the same reaction system. After optimizing the design, the sequences of the 4 pairs of specific primers are shown in Table 1.
[0040] Table 1. VNTR site amplification primers
[0041]
[0042] Taking the standard strain LT2 of Salmonella typhimurium as an example, the primers and ...
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The invention discloses a detection primer set and a kit for salmonella typhimurium MLVA typing, and applications of the detection primer set. The invention designs specific primers for four VNTR lociwith good resolving ability of salmonella typhimurium, establishes a multiplex PCR amplification detection method, sequences PCR amplification product fragments of the loci, uses a local Blast technology to analyze sequencing results and to obtain the combination of the number of repetitive sequences of the four loci, and performs MLVA typing on salmonella typhimurium. The method of the inventioncan perform stable typing detection on salmonella typhimurium, and obtains the number of VNTR repetitive sequences just by the analysis of the4 VNTR loci, the one-time multiplex PCR amplification detection, and the intuitive and convenient local Blast analysis. The resolution of the method is the same as that of an original MLVA-5 loci typing method, but the typing efficiency is higher than thatof the original MLVA-5 loci method, and the method of the invention is simpler and more convenient.
Description
Technical field: [0001] The invention belongs to the technical field of microbes, and in particular relates to a detection primer set, a kit and an application thereof for MLVA typing of Salmonella typhimurium. Background technique: [0002] Salmonella (Salmonella spp) is a zoonotic pathogen of great significance in public health. It can cause many serious diseases such as typhoid fever, paratyphoid fever, gastroenteritis, and sepsis in humans and animals, causing huge economic losses worldwide. At present, about 2610 Salmonella serotypes have been found in the world. Among them, Salmonella enterica subsp.enterica serovar Typhimurium (STM) is the dominant serotype existing in livestock, poultry and food, and also the main serotype causing human infection. [0003] Using effective typing methods to distinguish Salmonella from different sources, different serotypes and even different pathogenicity is of great significance for Salmonella hazard traceability, risk early warning...
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IPC IPC(8): C12Q1/689C12Q1/10C12N15/11C12R1/42
CPCC12Q1/689C12Q2600/16
Inventor 杨小鹃吴清平张菊梅曾海燕余树波王涓薛亮吴浩明张淑红
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY