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Vibrio cholerae typing and virulence gene detection kit and detection method

A detection kit and technology for Vibrio cholerae are applied in biochemical equipment and methods, material excitation analysis, and microbial determination/inspection, etc. Simple, easy to operate, high-throughput effect

Active Publication Date: 2011-02-09
DAAN GENE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Vibrio cholerae detection methods mainly include routine inspection methods, biological methods and molecular biological methods, but conventional inspection methods (direct microscopic examination and bacterial isolation and culture) and biological methods (various experimental animal models and cell methods) ) have disadvantages such as long detection time, small throughput or high condition requirements, and are not suitable for wide application

Method used

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  • Vibrio cholerae typing and virulence gene detection kit and detection method
  • Vibrio cholerae typing and virulence gene detection kit and detection method
  • Vibrio cholerae typing and virulence gene detection kit and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Vibrio cholerae typing and virulence gene detection kit and its use

[0049] 1. Prepare a kit comprising the following components:

[0050] DNA extraction solution (1.2 mL / tube) 1 tube: composed of 40 mM NaOH, 20 mM Tris-HCl (pH8.8), 5% TritonX-100, 0.1 mM EDTA (pH8.0).

[0051] PCR reaction solution (650μl / tube) 1 tube: PCR reaction buffer consists of Tris-HCl (50mmol / L, pH8.5), MgCl 2 (8mmol / L), KCl (250mmol / L) and dNTPs (25mmol / L); The four Vibrio cholerae oligonucleotide probe sequences used for fluorescent PCR amplification are shown in the sequence table SEQ.ID.NO9-12 respectively , the two ends of the SEQ ID NO9 probe are respectively combined with the fluorescence generating group FAM and the fluorescence quenching group BHQ1, and the two ends of the SEQ ID NO10 probe are respectively combined with the fluorescence generation group CY5 and the fluorescence quenching group BHQ2, SEQ The two ends of the ID NO11 probe are respectively combined with a fluorescent ...

Embodiment 2

[0080] Application of Vibrio cholerae typing and virulence gene detection kit to detect clinical samples

[0081] Select 2 cases to be detected as Vibrio cholerae negative through the isolation and culture method, and 2 cases are detected to be Vibrio cholerae O139-positive specimens through the isolation and culture method, and the nucleic acid extraction, PCR amplification and result analysis of the samples are carried out with reference to Example 1, and the negative results are carried out simultaneously. , Detection of positive quality control products.

[0082] Interpretation of test results:

[0083] The amplification curve of the negative quality control product is not S-shaped curve, and the Ct value is greater than 37, indicating that the amplification of Vibrio cholerae nucleic acid in the negative quality control product (see attached figure 2 ). The amplification curve of the positive quality control product is an S-shaped curve, and the Ct value is less than 3...

Embodiment 3

[0087] Application of Vibrio cholerae typing and virulence gene detection kit to detect clinical samples

[0088] Select 3 cases to be detected as Vibrio cholerae negative through the isolation and culture method, and 6 cases are detected to be Vibrio cholerae positive specimens through the isolation and culture method, and the nucleic acid extraction, PCR amplification and result analysis of the samples are carried out with reference to Example 1, and the negative and positive Testing of quality control products.

[0089] The detection result is explained with embodiment 2.

[0090] The amplification curves of 3 cases of Vibrio cholerae-negative specimens tested by the isolation and culture method did not show an S-shaped curve, and the Cts were all greater than 37, indicating that there was no amplification of Vibrio cholerae nucleic acid in the 3 samples (see attached Image 6 ).

[0091] 6 samples tested positive for Vibrio cholerae by isolation and culture method were t...

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Abstract

The invention relates to a vibrio cholerae typing and virulence gene detection kit and a vibrio cholerae typing and virulence gene detection method. The kit comprises DNA extracting solution, PCR reaction solution, a Taq enzyme system, a positive quality control product and a negative quality control product, wherein the PCR reaction solution consists of PCR reaction buffer, four pairs of forward and reverse primers for the specificity of vibrio cholerae and four probes for the specificity of the vibrio cholerae; the primers and probes are designed according to the specificity conservative areas of nucleic acid sequences of a hemolysin gene, an O-antigen gene of O139 type vibrio cholerae, the O-antigen gene of O1 type vibrio cholerae and the virulence gene of cholera toxin (CTX). The detection kit and the detection method ensure a reliable and stable result, are easy and fast to operate and can realize the typing of the vibrio cholerae and fast judgment about whether the vibrio cholerae has a virulence gene or not, thereby facilitating the tracing and detection of the public health-related events caused by the vibrio cholerae and facilitating the routine monitoring of the vibrio cholerae.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a Vibrio cholerae typing and virulence gene detection kit and a detection method thereof. Background technique [0002] Cholera is a severe infectious disease with diarrhea as the main symptom. So far, seven world pandemics have occurred. The seventh world pandemic of cholera, caused by Vibrio cholerae Eltor, began in 1961 and affected 140 countries and regions. More than 4 million cases were reported. According to the WHO expert meeting, there are about 5.5 million cases worldwide each year, among which Asia, Africa and Latin America are more serious, causing 100,000 deaths in Asia and 20,000 deaths in Africa. To this day, cholera remains one of the most dangerous infectious diseases. Therefore, early rapid and correct diagnosis is of great significance to the treatment and prevention of the spread of this disease. [0003] At present, Vibrio cholerae has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12Q1/68G01N21/64
CPCY02A50/30
Inventor 田桢干方筠韩晓辉陆晔张继伦张宏钱宇何宇平何琼董瑞华高秀洁陈华云
Owner DAAN GENE CO LTD
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