Multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for streptococcus suis type 2 virulence gene and detection method thereof

A technology of multiplex fluorescence quantification and detection kit, applied in the field of detection, can solve the problems of time-consuming operation, false negative and missed detection, low detection sensitivity, etc., and achieve the effect of biological safety guarantee, wide quantitative linear range, and increased stability.

Inactive Publication Date: 2021-06-11
HANBANG MEDICAL SCI & TECH HARBIN CITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the published methods for the detection of Streptococcus suis type 2 virulence genes are based on agarose gel electrophoresis, such as the patent application number: CN200710067487.5 and CN201010119005.8 The agarose gel electrophoresis detection all adopts multiplex PCR The post-agarose gel electrophoresis detection method has relatively low detection sensitivity, which may lead to false negatives and missed detections. However, due to the false positives caused by aerosol pollution in the experimental operation process, the virus content cannot be quantitatively detected.
Another example is the patent application numbers: CN200610109571.4 and CN200610109569.7 all adopt the method of single-fold fluorescent PCR to detect the mrp and epf genes of Streptococcus suis type 2 respectively, which cannot quantify the virus content, and the operation is time-consuming
Another example is the patent application number CN201610255090.8, which discloses a dual fluorescent quantitative PCR primer and kit for simultaneous detection of Streptococcus suis universal type and Streptococcus suis type 2, which is only for the detection of Streptococcus suis type 2, and cannot detect whether it contains or contains any A virulence gene, which cannot provide help for the epidemiological research of Streptococcus suis, let alone quantitative detection

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  • Multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for streptococcus suis type 2 virulence gene and detection method thereof
  • Multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for streptococcus suis type 2 virulence gene and detection method thereof
  • Multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for streptococcus suis type 2 virulence gene and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0072] The design, synthesis, screening and verification of embodiment 1 primer

[0073] The relatively conserved regions of the epf, mrp and sly genes of Streptococcus suis type 2 were selected as targets to be detected, and according to the design principles of primers and probes, two pairs of primers and probe combinations were designed for each gene and sent to Sangong Science and Technology Co., Ltd. Synthesized by Bioengineering (Shanghai) Co., Ltd., the corresponding primers and probes are as follows:

[0074]

[0075]

[0076] Plasmid DNA containing three target gene fragments of Streptococcus suis type 2 epf, mrp and sly was used as the initial concentration sample (concentration was about 3×10 11 copies / mL), sequentially dilute 8 gradients down 10 times as templates. The corresponding PCR reaction solution was prepared by using two sets of primer probes of the above three genes combined with other PCR reaction components, and then carried out fluorescent quant...

Embodiment 2

[0089] The assembly of embodiment 2 kit

[0090] The specification of the kit is 100 tests / box, and the specific components and specifications are shown in Table 2-1 below.

[0091] Table 2-1 SS-2 kit information

[0092]

[0093]

[0094] Wherein the SS-2 PCR reaction Buffer is a commercial Buffer purchased from Faipeng Bio, the final concentration of HEPES in the PCR reaction is 20mmol / L, the final concentration of (NH4)2SO4 is 10mmol / L, and the final concentration of KCl is 50mmol / L, the final concentration of sucrose is 25mmol / L, Mg 2+ The final concentration of 2.5mmol / L.

[0095] The SS-2 primer-probe mixture is a primer-probe mixture containing the 3 genes epf, mrp and sly determined in Example 1, and the final primer concentration of epf, mrp and sly in the 25 μL quantitative PCR reaction system is 50nmol / L, the corresponding probe concentrations are 100nmol / L, 160nmol / L and 80nmol / L, and the universal primer concentration is 400nmol / L.

[0096] The SS-2 enz...

Embodiment 3

[0099] The use of embodiment 3 kits

[0100] Reagent preparation: take the kit out of the refrigerator at -20°C±5°C and dissolve it at room temperature, vortex and mix the reagent components in the kit, follow the steps: 17.5 μL of SS-2 PCR reaction buffer for each reaction, SS-2 Mix 0.5 μL of enzyme system and 2 μL of SS-2 primer-probe mixture, vortex and mix well, briefly centrifuge and aliquot into PCR tubes, aliquot 20 μL / reaction and move to the sample loading area.

[0101] Adding samples: Take 5ul sample nucleic acid, standards of different concentrations, negative control and positive control respectively and add them to the PCR reaction tube, cap the PCR reaction tube tightly, vortex and mix well, and centrifuge briefly.

[0102] PCR amplification: The reaction conditions on the ABI7500 fluorescent quantitative PCR instrument are: 95°C for 5 minutes, then 95°C for 15 seconds, 58°C for 45 seconds, 5 cycles; 95°C for 15 seconds, 60°C for 45 seconds (fluorescence collect...

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Abstract

The invention relates to a multiplex fluorescent quantitative PCR detection kit for a streptococcus suis type 2 virulence gene and a detection method thereof. The multiplex fluorescent quantitative PCR detection kit mainly comprises an SS-2 primer probe mixed solution, a universal primer mixed solution, an SS-2 PCR reaction solution, an SS-2 primer probe mixed solution, an SS-2 negative control, an SS-2 positive control and an SS-2 quantitative standard substance. By adopting the kit, quantitative detection of extracellular factor epf, lysozyme release protein mrp and hemolysin sly genes of streptococcus suis type 2 virulence genes can be realized. The detection sensitivity of the kit is high, and the lowest detection limit is 1.0 * 10 < 3 > copies/mL; the quantitative linear range is wide, and three virulence genes of streptococcus suis type 2 of 1.0 * 10 < 3 > copies/mL-1.0 * 10 < 10 > copies/mL can be accurately quantified; the specificity of the kit is high, and no reaction with other bacteria and common porcine viruses exists; the repeatability is good, and the intra-batch and inter-batch variation coefficients are both smaller than 5%; the stability of the kit is high, the lowest detection limit reference of 1.0 * 10 < 3 > copies/mL can be detected by 100% after repeated freezing and thawing for 8 times, and the kit is very suitable for monitoring of conventional epidemic situations of swine diseases and detection requirements of biological products from swine blood on streptococcus suis.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a multiple fluorescent quantitative PCR detection kit and a detection method for Streptococcus suis type 2 virulence genes. Background technique [0002] Streptococcus suis is a common pathogen of pigs. Streptococcus suis is a general term for a class of diseases caused by various streptococci in pigs. According to the difference of capsular polysaccharides on the surface of the bacteria, it is divided into 35 serotypes (type 1-34) and 1 / 2), among which type 2 is the most common and the most pathogenic, followed by types 1, 7 and 9. Among them, the disease caused by Streptococcus suis type 2 is an important zoonotic disease. This pathogen can cause various diseases such as meningitis, arthritis, endocarditis, pneumonia, sepsis and even acute death in pigs. It can also infect humans and cause death. [0003] The virulence of Streptococcus suis is closely related to virulence f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/46
CPCC12Q1/686C12Q1/689C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 余美伦秦香芹朱佳伟耿东涛赵丽孟庆雪宫佳欣高丹何佳李常禄王磊王宁陶振明白雪峰
Owner HANBANG MEDICAL SCI & TECH HARBIN CITY
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