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Multiple-fluorescence PCR detection method and kit for S.hyicus exfoliative toxin types

A technology for Staphylococcus suis and detection kits, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/testing, and can solve the problems of little guiding significance, long time-consuming, poor sensitivity and specificity And other problems, to achieve the effect of high sensitivity, saving consumables, and low cost

Inactive Publication Date: 2015-06-03
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

[0007] Although the above methods have been popularized and applied, they all have defects such as long time-consuming, poor sensitivity and specificity, and have little guiding significance for actual production.

Method used

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  • Multiple-fluorescence PCR detection method and kit for S.hyicus exfoliative toxin types
  • Multiple-fluorescence PCR detection method and kit for S.hyicus exfoliative toxin types
  • Multiple-fluorescence PCR detection method and kit for S.hyicus exfoliative toxin types

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the specificity test of primer, probe

[0038] Using bioinformatics and biological software for designing primers and probes, the sequences of the four exfoliation toxins EXHA, EXHB, EXHC and EXHD genes of Staphylococcus suis in the existing database (GenBank numbers in NCBI are: JQ728534, EU259000, JQ728530, AF515456) After the comparative analysis, the primers and probes shown in Table 1 were designed to realize multiple fluorescent PCR detection of Staphylococcus suis shedding toxins EXHA, EXHB, EXHC and EXHD. Table 1 is the sequence of primers and probes of Staphylococcus suis exfoliated toxin.

[0039] Table 1 Sequences of primers and probes for the shedding toxin of Staphylococcus suis

[0040]

[0041]

[0042] The fluorescent probe information after the probe sequence is fluorescently labeled is shown in Table 2

[0043] Table 2

[0044] EXHA

EXHA-Prob-FAM

FAM-tgaagcgccttttggagcagga-BHQ

EXHB

EXHB-Prob-JOE

JOE-...

Embodiment 2

[0049] Embodiment 2: the specificity test of multiple fluorescent PCR detection reagent

[0050] Multiple detection reagents were prepared using the PCR buffer in the Premix Ex Taq kit of TaKaRa Company, wherein the amount of each primer added in Table 1 was 5-15 pmol, and the amount of various probes added in Table 1 was 2 -10pmol, 10μL of Premix solution (including TaKaRa Ex Taq0.5U, dNTPs and Ex Taq Buffer), add water to a total volume of 20μL, in which the volume of the DNA solution extracted from the detection sample is 2μL, and finally placed in a fluorescent quantitative PCR instrument for reaction, reaction conditions As follows: 95°C for 30 seconds, thermal cycle of 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles.

[0051] Single-positive templates (cultures of strains HZ-2, ExhB1-2007, ZC-2, and GZ-3, which produce shedding toxins EXHA, EXHB, EXHC, and EXHD, respectively) and quadruple-positive templates were used, respectively. (for an equal mix of cul...

Embodiment 3

[0056] Example 3: Sensitivity test of multiple fluorescent PCR detection reagents for Staphylococcus suis exfoliated toxin

[0057] In order to further test the sensitivity of the reagent, a 10-fold gradient dilution was carried out on the positive sample of Staphylococcus suis exfoliated toxin. 6 times, and then compared with multiplex and singlex PCR detection of Staphylococcus suis shedding toxin, the results are shown in Table 4. Table 4 is a data table of comparative experimental results of multiplex and singlex PCR detection reagents on 10-fold serially diluted positive samples.

[0058] Table 4

[0059]

[0060]

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Abstract

The invention discloses a multiple-fluorescence PCR detection method and a kit for four exfoliative toxin types (EXHA, EXHB, EXHC and EXHD) of S.hyicus, and belongs to the field of multiple-fluorescence PCR detection methods and kits. The method provided by the invention employs primers with sequences shown as SEQ ID NO: 1-8, and also employs Taqman probes with sequences shown as SEQ ID NO: 9-12. The invention also provides the multiple-fluorescence PCR detection kit for S.hyicus exfoliative toxin EXHA, EXHB, EXHC and EXHD. The kit comprises the above primer and the Taqman probes, and also comprises DNA polymerase, a PCR buffer solution, dNTP and water. The kit provided by the invention has the advantages of being strong in specificity, high in sensitivity, rapid, convenient to operate, capable of saving materials, low in cost and the like, is applicable to detection of the four exfoliative toxin types of S.hyicus, and is used to scientific research and clinical application.

Description

technical field [0001] The invention relates to a multiple fluorescent PCR method and a kit, in particular to a multiple fluorescent PCR method and a kit for typing four types of shedding toxins (EXHA, EXHB, EXHC and EXHD) of Staphylococcus suis. Background technique [0002] The shedding toxins produced by staphylococci can cause severe skin diseases in humans and animals. For example, Staphylococcus aureus can cause scalded skin syndrome in humans, and Staphylococcus suis can cause exudative dermatitis in piglets. There are 9 types of exfoliation toxins discovered so far, including ETA, ETB and ETD produced by Staphylococcus aureus, EXHA, EXHB, EXHC and EXHD produced by Staphylococcus suis, and two other toxin proteins of Staphylococcus suis discovered by Japanese researchers SHETA and SHETB. [0003] S. hyicus can cause large-scale exudative dermatitis (Exudative Epidermitis, EE) in pigs. It is an acute, highly contagious swine infectious disease. Sexual dermatitis is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/44
CPCC12Q1/686C12Q2537/143C12Q2561/101C12Q2563/107
Inventor 宋长绪蒋智勇张乐宜蔡汝健李艳刘燕玲
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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