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A kind of vibrio cholerae analysis typing kit

A technology of Vibrio cholerae and a kit is applied in the field of molecular typing detection kits for Vibrio cholerae, which can solve the problems of misjudgment and overlapping of amplification products, and achieve the effects of simple experimental operation and simple typing.

Active Publication Date: 2015-08-12
AGCU SCIENTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a kit for performing fluorescent labeling compound amplification and typing of Vibrio cholerae by using the VNTR locus, and to solve the problem of amplification caused by the relatively small allele range of the VNTR locus of Vibrio cholerae in the prior art. Increase product overlap and cause misjudgment

Method used

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  • A kind of vibrio cholerae analysis typing kit
  • A kind of vibrio cholerae analysis typing kit
  • A kind of vibrio cholerae analysis typing kit

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Experimental program
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Embodiment 1

[0029] The design of embodiment 1 kit and the determination of amplification conditions

[0030] 1. Selection of loci

[0031] Six VNTR loci of V. cholerae with high polymorphism were selected, and the relevant information of their alleles is shown in Table 3. The template sequence was downloaded, the locus sequence was downloaded from NCBI genebank, and the position of the sequence repeat region was determined. Allelic ranges were determined by aligning the sequences of different strains of V. cholerae at the same locus in Genebank. The result is as follows:

[0032] Table 4 loci and allele information

[0033] loci

Serial Genebank number

repeat location

allele range

VC1650

CP002555.1

1646667-1646729

2-14

VC0437

CP003330.1

466934-467328

2-24

VC0147

CP003330.1

137105-137159

2-17

VCA0171

CP003331.1

187738-187875

6-26

VCA0283

CP003331.1

303918-303989

3-30

...

Embodiment 2

[0085] The preparation of embodiment 2 allelic typing standards

[0086] The Vibrio cholerae strains collected by Zhejiang Provincial Center for Disease Control and Prevention were tested, and samples of the same locus and alleles of different sizes were selected. The following table shows the composition of alleles of each locus:

[0087] Table 14 Allele Information

[0088]

[0089] Amplify with VC1650, VC0437, VC0147, VCA0171, VCA0283 and VC1457 single locus amplification primers (primer concentration 10 μM) respectively. The genomic DNA sample can be bacterial liquid or extracted DNA.

[0090] The primer sequences are as follows:

[0091] Table 15 Primer Sequence

[0092]

[0093] A. The amplification system is as follows:

[0094] Table 16 Kit Configuration Composition

[0095]

[0096] The Reaction Mix mentioned in it is MgCl 2 7.5mM, Tris-HCl buffer125mM, KCl125mM, dNTPs7.5mM, BSA2mg / ml.

[0097] B. Amplification thermal cycle

[0098] Table 17 Amplific...

Embodiment 3

[0108] Embodiment 3: MLVA detection of 19 bacterial strains

[0109]The strains were derived from the Vibrio cholerae strains preserved by the Zhejiang Provincial Center for Disease Control and Prevention, and 19 strains collected at different times were randomly selected. These samples were classified into 12 types by the CDC using the classic PFGE method. Nos. 10-14 belonged to the same type, Nos. 15, 16, 17, and 19 belonged to the same type, and the other strains were the only type.

[0110] By the experimental system of embodiment 1, amplification procedure, detection method detects, and 19 strains typing results are as follows:

[0111] Table 18 strain typing results

[0112]

[0113] It can be seen from the results in the table that the 19 strains were divided into 17 types, among which the experimental numbers 12 and 13, and 17 and 19 were respectively the same type, showing better typing ability.

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Abstract

The invention relates to a vibrio cholera analysis typing kit. A fluorescently-marked composite primer is used for amplifying six VNTR (variable number of tandem repeats) loca of vibrio cholera respectively. According to the vibrio cholera analysis typing kit provided by the invention, the six loca are divided into three groups, and four fluorescent markers are involved in total; after capillary electrophoresis detection, by comparing with an allele typing standard substance, the type of vibrio cholera can be obtained quickly and accurately.

Description

technical field [0001] The present invention relates to a Vibrio cholerae molecular typing detection kit, in particular to a fluorescent marker compound amplification system and an allelic typing standard for simultaneous analysis of six Vibrio cholerae VNTR loci, the system can be applied Traceability of Vibrio cholerae. Background technique [0002] Bacterial diseases have always endangered human health and the healthy development of animal husbandry. With the improvement of living standards and the increasing frequency of population mobility, food safety problems caused by food-borne bacteria have become a social public health problem faced by the world. However, factors such as population growth, rapid development of science and technology, and popularization of interventional medical devices have made the incidence of nosocomial infection remain high, which has become the most difficult problem in clinical practice. [0003] The identification and typing technology of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
CPCC12Q1/04C12Q1/6858C12Q2545/113Y02A50/30
Inventor 葛斌文罗芸卢青金大智张政周丽萍葛海鹏郭育林
Owner AGCU SCIENTECH
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