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Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis

A Streptococcus suis and dual fluorescence technology, which is applied in the field of nucleic acid detection, can solve the problems of simultaneous detection of Streptococcus suis and Streptococcus suis type 2, increase the workload and cost of epidemic monitoring, and shorten the detection time and cost. Experimental time and cost, the effect of high sensitivity

Active Publication Date: 2016-07-20
SHAOGUAN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, researchers have established a number of fluorescent PCR methods for Streptococcus suis, including the general-purpose fluorescent PCR method designed by Gao Zhiqiang for the 16SRNA of Streptococcus suis, and the fluorescent PCR method for Streptococcus suis type 2 designed by Gao Zhiqiang and Sun Yang for the cps2J gene, but these The method is single fluorescent PCR, which cannot realize the simultaneous detection of Streptococcus suis and Streptococcus suis type 2 in a single tube, which increases the workload and cost of epidemic monitoring

Method used

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  • Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis
  • Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis
  • Dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting general type and type 2 Streptococcus suis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Simultaneous detection of dual fluorescent quantitative PCR primers and probes for Streptococcus suis universal type and Streptococcus suis type 2

[0056] According to the nucleic acid sequences of Streptococcus suis universal type and Streptococcus suis type 2, the present invention is designed to simultaneously detect dual fluorescent quantitative PCR primers and probes for both Streptococcus suis universal type and Streptococcus suis type 2; A large number of primers and probes were screened, and a set of primers and probe sequences with high sensitivity and specificity were screened out. The sequences are as follows:

[0057] Primer GDH-F: 5'-GAGCTCTTCTCTACACTTGAGCC-3' (SEQ ID NO: 1),

[0058] Primer GDH-R: 5'-CCATGGAACACGGAAGCTG-3' (SEQ ID NO: 2),

[0059] Probe GDH-P: 5'-FAM-TTGAAGCACACCCAGAATACATCGAAGAA-TAMRA-3' (SEQ ID NO: 3),

[0060] Primer CPS2J-F: 5'-GATAGATGACGGTTTCTTCAGATTCAT-3' (SEQ ID NO: 4),

[0061] Primer CPS2J-R: 5'-CCATTTGGTAACCGGAAAAG...

Embodiment 2

[0063] Example 2 optimizes the optimization of double fluorescent quantitative PCR reaction conditions

[0064] ABI7500 fluorescent quantitative PCR system was used for amplification and analysis, and PremixExTaq (ProbeqPCR) was used as the reaction buffer. First, optimize the annealing temperature under the reaction system recommended by the company, set 6 annealing temperatures, respectively 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, and the reaction condition is 95°C / 5min for pre-denaturation And start the Taq enzyme, then denature at 95°C / 15s, anneal and extend at 50-60°C for 30s and collect fluorescence, a total of 40 cycles. After comparing the results of the same sample at 6 different annealing temperatures, it was found that the results at 56°C were slightly better than other annealing temperatures, that is, 56°C is the optimal annealing temperature for dual fluorescent quantitative PCR.

[0065] After determining the annealing temperature, optimize the reaction concentratio...

Embodiment 3

[0068] Example 3 Positive standard preparation, two-color fluorescent quantitative PCR amplification and standard curve drawing

[0069] 1) Preparation of standard samples

[0070] In order to draw the standard curve of dual fluorescence PCR, we extracted the positive Streptococcus suis universal type and Streptococcus suis type 2 genomic DNA as PCR templates respectively, and amplified with the following designed primer pairs to construct positive standard DNA. The primer sequences are:

[0071] GDH-SF: 5'-GGAATTCCATATGTCAAATGCC-3' (SEQ ID NO: 7),

[0072] GDH-SR: 5'-CCATGGAACACGGAAGCTG-3' (SEQ ID NO: 8),

[0073] The length of the amplified product of the primer pair is 214bp;

[0074] CPS2J-SF: 5'-AGGAATTCCATATGGAAAAAGTCAGCATTATTG-3' (SEQ ID NO: 9),

[0075] CPS2J-SR: 5'-CCGCTCGAGTTAATCATTATTTTTTTCTTCCC-3' (SEQ ID NO: 10),

[0076] The length of the amplified product of the primer pair is 1019bp.

[0077] The resulting amplified product was separated by 1% agarose elec...

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Abstract

The invention discloses a dual-fluorescence quantitative PCR (Polymerase Chain Reaction) primer, kit and method for simultaneously detecting the general type and type 2 Streptococcus suis. The invention breaks through the limitation of the common PCR method, and can quantify the bacterial content in a sample in real time, has high sensitivity, strong specificity, good repeatability and obvious clinical diagnosis effects, the lowest detection limit reaches 5 copies per reaction; the Streptococcus suis and the type 2 Streptococcus suis can be specifically recognized, no response is generated to other bacteria; the variable coefficient of repeated detection is less than 1.19%; furthermore, the dual-fluorescence quantitative PCR primer for simultaneously detecting the general type and type 2 Streptococcus suis and the kit and method thereof disclosed by the invention are very suitable for detection of a large number of clinical samples and the routine epidemic surveillance of the Streptococcus suis, and extremely high in application values.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and relates to a dual fluorescence quantitative PCR primer, a kit and a method for simultaneously detecting Streptococcus suis universal type and Streptococcus suis type 2. Background technique [0002] Streptococcus suis is one of the main pathogens that cause significant economic losses in the pig breeding industry. Streptococcus suis exists in almost 100% of large-scale pig farms around the world, and can be transmitted through the respiratory tract, mainly causing sepsis, meningitis, arthritis, pneumonia, Symptoms such as endocarditis. At the same time, Streptococcus suis is also a zoonotic pathogen. People can be infected by contact with infected pigs or pork products, causing sepsis, meningitis, and even shock-like syndrome leading to death. Although most of the people infected with Streptococcus suis were sporadic cases of occupational contact in the past 50 years, such as farmers, v...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/113
Inventor 吴静波南文金彭国良黄健强胡鸿惠
Owner SHAOGUAN COLLEGE
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