LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof

A Vibrio cholerae and kit technology, applied in the biological field, can solve the problems of affecting PCR amplification results, difficult to meet rapid identification, expensive and other problems, and achieve the effects of shortening reaction time, improving detection sensitivity, and short time consumption

Inactive Publication Date: 2014-07-30
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the method of detecting pathogenic microorganisms in food is mainly based on the traditional method, that is, the separation and identification method. This method takes a long time, usually 5-7 days, sometimes 10-15 days, and it is difficult to meet the requirements of rapid identification. Need; the PCR technology developed in recent years is a fast, sensitive, and specific technology, but at present this technology still relies on the pre-enrichment step of the traditional method, and the enrichment solution often contains PCR inhibitors, thereby Affect the amplification result of PCR; rapid ELISA method detection, as a screening method, has the characteristics of fast and sensitive, and is widely welcomed as a screening method, but the kits used are all from abroad, the price is expensive, and it needs to be equipped with its special instrument

Method used

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  • LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof
  • LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof
  • LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of immunomagnetic beads coupled with anti-Vibrio cholerae monoclonal antibody

[0044] Obtaining monoclonal antibody against Vibrio cholerae

[0045] The present invention can use the anti-Vibrio cholerae monoclonal antibody that has been disclosed in the prior art. In order to achieve a better detection effect, preferably, the present invention uses the cholera antibody secreted by the hybridoma cell line with the deposit number of CGMCC No.6754. Vibrio flagellin monoclonal antibody, the specific information and obtaining method of the monoclonal antibody have been described in detail in Chinese invention patent application 201210532294.3. In order to more fully disclose the above content, the present invention provides a specific acquisition method here:

[0046] 1. Cell culture and extraction of flagellin

[0047] Vibrio cholerae (Vibrio cholera) (VC-75, isolated and stored in the laboratory) was inoculated in T1N1 medium (purchased from Beij...

Embodiment 2

[0103] Embodiment 2: the composition of the LAMP detection kit of Vibrio cholerae

[0104] (1) Immunomagnetic beads coupled with anti-Vibrio cholerae monoclonal antibody; for example, the immunomagnetic beads prepared in Example 1;

[0105] (2) LAMP reaction solution, which includes the outer upstream primer (F3) shown in the sequence table SEQ ID No.1, the outer downstream primer (B3) shown in the sequence table SEQ ID No.2, and the sequence table SEQ ID No.3 The inner upstream primer (FIP) shown in the sequence table, the inner downstream primer (BIP) shown in SEQ ID No.4 in the sequence table, the circular upstream primer (LF) shown in SEQ ID No.5 in the sequence table, and the sequence table SEQ ID No. .6 The circular downstream primer (LB); said primers were all synthesized by Dalian Bao Biological Engineering Co., Ltd.;

[0106] Further, the LAMP reaction solution also includes ThermoPol buffer, dNTPs, betaine and MgSO 4 ; Wherein ThermoPol buffer, purchased from NEB C...

Embodiment 3

[0111] Embodiment 3: detect the specificity and sensitivity test of vibrio cholerae kit

[0112] 1. Assay Specificity Analysis

[0113] The strains listed in Table 3 were detected by IMS-LAMP respectively, and the results showed that the amplified products of Vibrio cholerae standard strains were fluorescent green after staining with SYBR Green Ⅰ, and a typical ladder-shaped amplification band was detected by agarose gel electrophoresis ; while the rest of the strains had no specific amplification (see figure 1 ), without any false positive and false negative results, indicating that this method has good specificity for detecting Vibrio cholerae.

[0114] Table 3 list of experimental strains

[0115] serial number

strain name

Numbering

1

Vibrio cholerae

isolate VC-75

2

Vibrio alginolyticus

ATCC1833

3

Vibrio vulnificus

ATCC1758

4

Vibrio river

ATCC1.1611

5

Vibrio venice

ATCC1.1612

...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and a detection method thereof and specifically relates to a group of primers for detecting the vibrio cholerae, which have oligonucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 6 in a sequence table, as well as the kit containing the primers and the detection method thereof. The kit disclosed by the invention is high in sensitivity, strong in specificity, low in cost and simple and convenient to operate.

Description

technical field [0001] The invention relates to a LAMP detection kit for Vibrio cholerae and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Cholera is a severe intestinal infectious disease caused by Vibrio cholera (VC). The disease has an acute onset, rapid spread, and wide coverage. It is an international quarantine infectious disease and is listed as one of the Class A infectious diseases in my country. , is one of the main foodborne pathogenic microorganisms, which always threatens and brings harm to human beings. Human beings are the only susceptible persons to Vibrio cholerae under natural circumstances, and they are mainly transmitted orally through contaminated water or food. Under certain conditions, after Vibrio cholerae enters the small intestine, it relies on the movement of flagella to pass through the mucus layer on the surface of the mucosa, adheres to the epithelial cells of the intestinal wall through the a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/63
CPCY02A50/30
Inventor 曾静张蕾程晋霞张西萌魏海燕张琳
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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