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Cross isothermal amplification primer groups for detecting streptococcus suis, kit and application

A technology for isothermal amplification of Streptococcus suis, applied in the biological field, can solve problems such as hindering application and popularization, difficulty in judging results, time-consuming and labor-intensive problems, and achieve intuitive and accurate reaction results, easy reaction results, and low cost

Inactive Publication Date: 2017-11-28
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used SS detection methods mainly include bacterial isolation, serological methods and PCR technology, among which bacterial isolation and serological methods are not only time-consuming and laborious, but also the results are not easy to judge
Although PCR technology can achieve relatively fast detection of SS, it requires expensive thermal cyclers and skilled operators during the amplification process, which hinders the widespread application and promotion of this technology in veterinary grassroots

Method used

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  • Cross isothermal amplification primer groups for detecting streptococcus suis, kit and application
  • Cross isothermal amplification primer groups for detecting streptococcus suis, kit and application
  • Cross isothermal amplification primer groups for detecting streptococcus suis, kit and application

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Embodiment 1

[0043] Design and synthesis of embodiment 1 cross primer constant temperature amplification primer

[0044] 1. Primer design

[0045] According to the principle of primer design, aiming at the conserved region sequence of the recN gene of Streptococcus suis, use Priemer5 software to design primers. According to our experience, the GC content of the primers is guaranteed to be between 40% and 60%, and the Tm value of each primer is between 50 and 60°C. about. Then we used the PrimerSelect tool of Larsergene7.0 biological software to preliminarily screen the primers. The screening principle was to ensure that the dG value between each primer pair was small. The primer sequences are shown in Table 1 (B2 and B1 primers are not biomarked at this time), the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and the synthesized primers were diluted with sterilized three-distilled water to a concentration of 10 μM, - Store at 20°C.

[0046] Tab...

Embodiment 2

[0049] Application of Example 2 Cross Primer Constant Temperature Amplification Primer in Detecting Streptococcus suis

[0050] 1. Extraction of Streptococcus suis genomic DNA

[0051] (1) Get the serum type 2 Streptococcus suis preserved in glycerol, PBS (pH value is 7.4, concentration is 0.15M phosphate buffer saline: KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O 2.9 g, NaCl 8 g, KCl 0.2 g) were dissolved, 50 μL was inoculated into 5 mL TSB medium, and cultured with shaking at 37 °C for 24 h.

[0052] (2) Take one loop of the inoculation loop and streak it on the TSA agar medium plate, at 37°C, 5% CO 2 Conditioned for 24h.

[0053] (3) A single colony was picked, inoculated in 5 mL TSB liquid medium, and cultured with shaking at 37°C for 12 hours.

[0054] (4) Take 1 mL of the bacterial culture and centrifuge at 4000 r / min for 5 min to collect the bacteria.

[0055] (5) Wash 3 times with 500 μL sterilized ultrapure water.

[0056] (6) Resuspend the pellet in 100 μL of sterili...

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Abstract

The invention discloses cross isothermal amplification primer groups for detecting streptococcus suis, a kit and application. The primer groups comprise primer groups with nucleotide sequences shown as SEQ ID NO.1-5. The kit comprises the primer groups and a nucleic acid test strip. The using method of the kit comprises the following steps: preparing a cross isothermal amplification reaction system; and detecting the reacted product by using the nucleic acid test strip and directly reading, wherein the positive result is that two stripes appear, one stripe is positioned in a detection zone, and the other stripe is positioned in a quality control zone. The kit is simple in operation and low in cost, the reaction result is easy to observe, the specificity is high, the kit is very applicable to export quarantine, food sanitation and field detection of livestock farms, and large-scale popularization and application can be easily realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cross isothermal amplification primer set, kit and application for detecting Streptococcus suis. Background technique [0002] Streptococcus suis (SS) can cause meningitis, arthritis, septicemia, endocarditis, multiple serositis and bronchopneumonia in pigs, and the mortality rate of pigs can reach 80%. SS is divided into 35 serotypes, of which 1, 1 / 2, 2, 7, 9, and 14 are the main pathogenic serotypes. SS is an important zoonotic pathogen. Human infection with SS can lead to sepsis, meningitis, endocarditis and even death, which has aroused great concern in public health. [0003] At present, commonly used SS detection methods mainly include bacterial isolation, serological methods and PCR technology, among which bacterial isolation and serological methods are not only time-consuming and laborious, but also difficult to determine the results. Although PCR technology c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/46
CPCC12Q1/6844C12Q1/689C12Q2565/625C12Q2563/101
Inventor 勾红潮李春玲蔡汝健宋帅李淼蒋智勇李艳杨冬霞楚品品
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI