Recombinase polymerase amplification (RPA) method, RPA special primers and RPA probe for detecting streptococcus suis serotype 2 as well as application of RPA method, RPA special primers and RPA probe

A Streptococcus suis and probe technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as inability to detect quickly, and achieve the effects of saving detection time, simple method and high sensitivity

Inactive Publication Date: 2018-08-10
李佳萌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires specific equipment, time-consuming, and complex processing of samples. Once an epidemic occurs, it cannot be detected quickly

Method used

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  • Recombinase polymerase amplification (RPA) method, RPA special primers and RPA probe for detecting streptococcus suis serotype 2 as well as application of RPA method, RPA special primers and RPA probe
  • Recombinase polymerase amplification (RPA) method, RPA special primers and RPA probe for detecting streptococcus suis serotype 2 as well as application of RPA method, RPA special primers and RPA probe
  • Recombinase polymerase amplification (RPA) method, RPA special primers and RPA probe for detecting streptococcus suis serotype 2 as well as application of RPA method, RPA special primers and RPA probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Design and screening of embodiment 1, type 2 Streptococcus suis RPA detection primers and probes

[0041] (1) Design of primers and probes

[0042] The inventor analyzed and determined through literature search that the specific sequence in the cps2J gene of Streptococcus suis type 2 used in the present invention is the target gene. The known template gene sequence was obtained from the NCBI database, i.e. the nucleotide sequence shown in SEQ ID NO.1, and the above sequence was synthesized by Nanjing KingScript Biotechnology Co., Ltd. It can be used as a template in the process of needle screening and optimization of reaction system. According to the design principles of RPA primers and probes, 5 primers and 1 probe were designed, as shown in Table 1.

[0043] Table 1 Primer and probe sequences

[0044]

[0045] (2) Primer screening

[0046] Artificially synthesize a positive plasmid containing the sequence shown in SEQ ID NO.1 of the cps2J gene of Streptococcus ...

Embodiment 2

[0055] Embodiment 2: Optimization of RPA reaction system, amplification and detection conditions

[0056] In the process of primer screening, there are still false positives in the detection of lateral flow nucleic acid detection test strips, so the RPA reaction system, amplification and detection conditions need to be optimized

[0057] (1) Primer probe concentration

[0058] Set the concentration gradient of the reverse primer as 10 μmol / L, 5 μmol / L, and 2.5 μmol / L, and the concentration of the probe as 10 μmol / L, 5 μmol / L, and 2.5 μmol / L. The primers were combined with the probes of three concentrations to form 9 combinations, and a set of negative controls was set for each combination. RPA amplification was carried out at 37°C, and the color development of the detection line of the lateral flow nucleic acid detection strip was used as an indicator for the completion of the amplification. The combination with the best amplification effect and no false positives was select...

Embodiment 3

[0067] Embodiment 3: Detection limit evaluation of RPA detection

[0068] Dilute the type 2 Streptococcus suis positive plasmid into 10-fold ratio 4 - 10 0 For a series of different concentrations such as copy / μL, 1 μL was added to the reaction system determined in Example 2, and the primer combination screened out was used to perform RPA detection on the templates with different copy numbers above using the reaction conditions determined in Example 2. Observe the detection limit of the RPA assay.

[0069] Results (see Figure 4 ), from 10 2 Copy / μL begins above sample all to be positive, and negative control is negative, illustrates that the detection limit of RPA detection method of the present invention is 10 2 copy / react.

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Abstract

The invention provides a recombinase polymerase amplification (RPA) detection method, RPA special primers and a RPA probe for detecting streptococcus suis serotype 2 as well as application of the RPAmethod, the RPA special primers and RPA probe in detection of the streptococcus suis serotype 2. The detection method, the special primers and the probe are designed based on streptococcus suis cps2Jconserved sequence design, and have oligonucleotide sequences shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. An RPA technology is firstly applied to the detection of the streptococcus suis serotype 2; the method simulates an enzymatic reaction process of DNA replication in vivo, depends on a specific enzyme and protein combination including recombinase, single-stranded binding protein and DNA polymerase to amplify a DNA template, and realizes the amplification of the specific nucleic acid sequence at a constant temperature of 37 DEG C; the amplified product is visually distinguished bymeans of a lateral chromatographic test strip. The detection method established by the invention is high in sensitivity, and has a detection limit reaching up to 102 copies/reaction; the special primers are strong in specificity, and has no cross reaction with other pathogens; the requirement for hardware equipment is low, and the reaction time is only 15min; a sample does not need to be subjectedto complicated treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to the molecular biology of type 2 Streptococcus suis, relates to a method for detecting type 2 Streptococcus suis and its application, and particularly relates to a rapid The method for detecting type 2 Streptococcus suis, its special primers and probes and its application. Background technique [0002] Streptococcus suis, S.s uis), belonging to the genus Streptococcus of the family Bacillus, Lactobacillus, and Streptococcus. Streptococcus suis is a zoonotic pathogen that can cause meningitis, arthritis and septicemia in pigs, and can also cause infections in humans, leading to meningitis and toxic shock syndrome. It was first confirmed in Guangdong Province in 1991 in my country The existence of Streptococcus suis disease, and in Jiangsu and Sichuan in 1998 and 2005 respectively broke out the public health events of large-scale type 2 Streptococcus suis epidemic infecting pigs and humans. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/689C12Q1/14C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2521/101C12Q2521/507C12Q2563/107
Inventor 齐永李越希李晓玲李佳萌饶继先沈万鹏曾雯雯刘苏云林煜郑书龙邓艺郦钰超
Owner 李佳萌
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