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Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application

A real-time fluorescence quantification, Streptococcus equi technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, resistance to vector-borne diseases, etc., can solve the problems of low sensitivity, undetectable, time-consuming and costly, and achieve specificity Good results

Active Publication Date: 2022-07-12
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods have relatively low sensitivity and cannot be detected when the antibody level is relatively low; and require professionals to operate, which is time-consuming and costly, and can only be used for laboratory diagnosis, not suitable for a large number of samples Detection; the multiplex PCR detection technology established in the existing molecular biology diagnosis can only distinguish Streptococcus equi subspecies from other Streptococcus suis and its sensitivity is relatively low

Method used

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  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application
  • Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Design and synthesis of specific amplification primer pairs

[0028] In this example, the pgm gene was amplified, and the analysis and alignment were performed according to the pgm gene sequence of SEZ published in GenBank (accession number: CP002904.1). Design a pair of specific primers (SEQ ID No. 1-2), upstream primer qPGM-F: 5'-CCCCGTAGCTCTCTGCAAT-3', downstream primer qPGM-R: 5'-GCAGACTGGGACGCTACC-3', the amplified fragment length is 89bp . The above primer pairs were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0029] The construction of embodiment 2 positive standard

[0030] In this example, the DNA of SEZ (C55138 strain was purchased from China Veterinary Drug Administration) was extracted with Mini BEST ViralRNA / DNA Extraction Kit, and its pgm gene was amplified by ordinary PCR. The reaction system is 25 μL: 12.5 μL of 2×PremixTap, 1.0 μL (10 μmol / L) of each of the upstream and downstream primers described in Example 1, 1.0 μL of template, ddH 2 O 7.5 μL. The amplification program was 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and 72 °C for 5 min. PCR amplification products were electrophoresed on 1.0% agarose. Then, according to the instructions of the DNA Purification Kit, the PCR product was recovered and purified, and the purified PCR product was ligated with the pMD19-T vector through Solution I ligase, and then the ligated product was thermally transformed into DH5α E. coli competent cells. Amp+ The positive clone obtained by plate scr...

Embodiment 3

[0032] Example 3 Establishment of the standard curve of fluorescence quantitative PCR

[0033] The recombinant plasmid pMD19T-SEZ was serially diluted to obtain 3.87×10 2 copies / μL~3.87×10 9 copies / μL of 8 dilutions of the standard as the reaction template. Three replicates were set for each dilution gradient and a negative control was established. Fluorescence quantitative PCR reaction system is 20 μL: SYBR GreenⅡMix (2×) 10 μL, upstream and downstream primers (SEQID No.1-2) each 0.6 μL (10 μmol / L), template 2 μL, ddH 2 O 6.8 μL. The reaction conditions were: (1) Pre-denaturation: 95°C, 30s, 4.4°C / s, 1 cycle; (2) PCR reaction: 95°C, 5s, 4.4°C / s; 54°C, 30s, 2.2°C / s; 72°C, 30s, 4.4°C / s, a total of 40 cycles. (3) Dissolution curve: 95°C, 5s, 4.4°C / s; 60°C, 1min, 2.2°C / s; 95°C, 0s, 0.11°C / s.

[0034] Taking the logarithm of the copy number of the standard as the X-axis and the ΔCT value as the Y-axis, draw the standard curve as follows figure 2 As shown, the standard curv...

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Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application. The nucleotide sequence of the real-time fluorescent quantitative PCR detection primer for streptococcus equi subsp. Zooepidemicus disclosed by the invention is as shown in SEQ ID No.1-2. The primer is used for performing fluorescent quantitative PCR detection on streptococcus equi subsp. Zooepidemicus, is good in specificity, relatively high in sensitivity, low in cost and rapid, and can be used for detecting a large number of samples.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to real-time fluorescent quantitative PCR detection primers, methods and applications of Streptococcus equi subsp. zooepidemicus. Background technique [0002] Streptococcus equi ssp. Zooepidemicus (SEZ) is one of the main pathogens of swine streptococcosis. It belongs to the group C streptococcus of the Langerhans group, and it is positive. The bacteria mainly causes horses, pigs, cattle, and dogs. , cats and other animals lower respiratory tract infection, and cause sepsis, meningitis, arthritis and other symptoms, and can cause sudden death in severe cases. [0003] SEZ has a wide range of infections and a high mortality rate. In addition to being infectious to pigs, the bacteria can also infect poultry through fecal transmission, resulting in a decrease in the egg production rate of poultry and sudden death in severe cases. It can be seen that SEZ is extremely harmful, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/14C12Q1/06C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 徐建琦付强余肇锋胡俊冶张鹏举张熙马春全
Owner FOSHAN UNIVERSITY
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