Fluorescent quantitative PCR method for simultaneous detection of streptococcus suis subtype 2 (SS2) extracellular protein factor and hemolysin gene

A fluorescent quantitative, extracellular protein technology, applied in the field of nucleic acid detection, to achieve the effects of short detection time, strong reagent versatility, avoiding pollution and dispersing toxins

Inactive Publication Date: 2013-02-13
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Taqman probe method has strong specificity, and needs to design specific fluorescent probes for different templates, which is expensive

Method used

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  • Fluorescent quantitative PCR method for simultaneous detection of streptococcus suis subtype 2 (SS2) extracellular protein factor and hemolysin gene
  • Fluorescent quantitative PCR method for simultaneous detection of streptococcus suis subtype 2 (SS2) extracellular protein factor and hemolysin gene
  • Fluorescent quantitative PCR method for simultaneous detection of streptococcus suis subtype 2 (SS2) extracellular protein factor and hemolysin gene

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Experimental program
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Effect test

Embodiment 1

[0029] According to the EF and SLY gene sequences of Streptococcus suis type 2 published in GenBank, the conserved sequences were selected through gene sequence comparison, and the GeneBank accession numbers of the selected EF and SLY public sequences were DQ417121.1 and DQ443530.1, respectively. EF and SLY primers were designed by Primer PremierS 5.0 and Oligo 6 software, and the primers were synthesized by Shanghai Sangon Biotechnology Company.

[0030] The upstream primer of EF is: 5'-GAAGAAGAACCCAAGGAACC-3'; the downstream primer of EF is: 5'-ACATTCTGACCACTCGCATC-3'. The size of the amplified EF fragment is 158bp, and the Tm value of the product is 84°C.

[0031] The upstream primer of SLY is: 5'-TTCCGATTTCGTATTCAACC-3'; the downstream primer of SLY is: 5'-AACTGTTTCCACCACTCCC-3'. The size of the amplified SLY fragment is 338bp, and the Tm value of the product is 80°C.

[0032] The extraction of embodiment 2 bacterial DNA

Embodiment 2

[0033] Inoculate sterilized Todd-Hewitt broth medium with a single colony of Streptococcus suis type 2 HA9801 strain, culture overnight at 37°C, take 1 mL of the bacterial solution, centrifuge at 12,000 rpm for 2 min, and resuspend the precipitate with 567 μL of TE buffer (pH 8.0). Add 30 μL of 10% SDS and 3 μL of 20 mg / mL proteinase K, mix well, bathe in 37°C water for 1 hour, add 100 μL of 5mol / L NaCl, mix well, then add 80 μL CTAB / NaCl solution, mix well, bathe in water at 65°C for 10 min , add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and mix evenly, centrifuge at 12000rpm for 10min, absorb the supernatant and add an equal volume of chloroform:isoamylalcohol (24:1) and mix evenly, centrifuge at 12000rpm for 10min, Aspirate the supernatant, add 0.6 volume of isopropanol, place at -20°C for 20 minutes to fully precipitate the DNA, centrifuge at 12,000 rpm for 10 minutes, wash the precipitate twice with 70% ethanol, and then dry it at room temperature to...

Embodiment 3

[0036] Fluorescent PCR reaction system:

[0037] The fluorescent quantitative PCR reaction system of the present invention is a 20ul system, operated on ice, and the following reagents are added in order:

[0038]

[0039] Among them, Master Mix is ​​SYBR Green I dye, polymerase, dNTP, Mg 2+ , PCR buffer mixture. Fluorescent PCR reaction conditions:

[0040] Pre-denaturation: 95°C, 5min

[0041] Fluorescent PCR amplification for 40-45 cycles: denaturation at 95°C for 10s; annealing at 55°C for 20s; extension at 72°C for 30s

[0042] Melting curve reaction program: 95°C, 5s; 65°C, 1min; 97°C, continuous; heating rate 2.2°C / s, start collecting fluorescence signals continuously at 65°C until 97°C.

[0043] Cooling: 40°C, 10s

[0044] Fluorescent PCR reaction criteria:

[0045] According to the Ct value and Tm value, the detection results of Streptococcus suis type 2 extracellular protein factor and hemolysin gene were judged.

[0046] The criteria for judging the amplif...

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Abstract

The invention relates to a fluorescent quantitative PCR method for simultaneously detecting type 2 extracellular protein factor EF and hemolysin gene SLY of Streptococcus suis based on a SYBR Green dye melting curve analysis method. According to the conserved sequences of EF and SLY genes of Streptococcus suis type 2 published in GenBank, two pairs of specific primers were designed and synthesized, the PCR reaction system and reaction conditions were optimized, and the simultaneous detection of Streptococcus suis type 2 was established by analyzing the amplification curve and melting curve SYBR GreenI dye fluorescence quantitative PCR method for EF and SLY genes. On this basis, the molar ratio of EF and SLY primers was optimized, and the amplification efficiency of the two pairs of primers was adjusted, so that the peak sizes of the two products in the melting curve were close. The EF and SLY genes of Streptococcus suis type 2 can be detected conveniently and simultaneously by using the established SYBR Green dye melting curve analysis method.

Description

technical field [0001] The invention relates to a multiple fluorescence quantitative PCR method for detecting the extracellular protein factor and hemolysin gene of Streptococcus suis type 2 by a SYBR Green dye dissolution curve analysis method, belonging to the field of nucleic acid detection. technical background [0002] Streptococcus suis (SS) is an important zoonotic pathogen with a worldwide distribution, and it is one of the main infectious diseases that have plagued the pig industry for many years. Acute infection of pigs often manifests as hemorrhagic septicemia and meningitis, and chronic infection manifests as arthritis, endocarditis, and suppurative lymph nodes. Toxic shock and meningitis in humans infected with Streptococcus. Streptococcus suis not only seriously hinders the healthy development of pig industry, but also threatens the safety of human life. Therefore, it is necessary to closely monitor the epidemiological changes of Streptococcus suis. [0003]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/46
Inventor 颜世敢朱丽萍陆承平陈正涛崔道石张秀美胡北侠许传田杨少华张琳
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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