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

A hemolytic Vibrio and detection kit technology, applied in the biological field, can solve the problems of affecting PCR amplification results, difficulty in satisfying rapid identification, and high price, so as to shorten reaction time, improve detection sensitivity, and save time Effect

Inactive Publication Date: 2014-11-19
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 parahaemolyticus and detection method thereof
  • LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof
  • LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] Obtaining monoclonal antibody against Vibrio parahaemolyticus

[0045] The present invention can use the anti-Vibrio parahaemolyticus monoclonal antibody that has been disclosed in the prior art. In order to achieve a better detection effect, preferably, the present invention uses the hybridoma cell line secreted by the preservation number as CGMCC No.6061. Parahaemolyticus Vibrio flagellin monoclonal antibody, the specific information and obtaining method of the monoclonal antibody can be found in the Chinese invention patent application CN102659942A.

[0046] Preparation of immunomagnetic beads coupled with the anti-Vibrio parahaemolyticus monoclonal antibody

[0047] 1. Preparation of Magnetic Nanobeads

[0048] The preparation method of the immunomagnetic beads of the present invention includes the preparation of nano-superparamagnetic Fe3O4 and th...

Embodiment 2

[0061] Embodiment 2: the composition of the LAMP detection kit of vibrio parahaemolyticus

[0062] (1) Immunomagnetic beads coupled with anti-Vibrio parahaemolyticus monoclonal antibody; for example, the immunomagnetic beads prepared in Example 1;

[0063] (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.;

[0064] Further, the LAMP reaction solution also includes ThermoPol buffer, dNTPs and MgSO 4 ; Wherein ThermoPol buffer, purchased fro...

Embodiment 3

[0069] Embodiment 3: detect the specificity and sensitivity test of Vibrio parahaemolyticus kit

[0070] 1. Assay Specificity Analysis

[0071] The strains listed in Table 3 were detected by IMS-LAMP respectively, and the results showed that the amplified product of the standard strain of Vibrio parahaemolyticus showed fluorescent green staining by SYBR GreenⅠ, and a typical ladder-like amplification was detected by agarose gel electrophoresis band; 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 parahaemolyticus.

[0072] Table 3 list of experimental strains

[0073] serial number

strain name

Numbering

1

Vibrio parahaemolyticus

ATCC17802

2

Vibrio alginolyticus

ATCC1833

3

Vibrio vulnificus

ATCC1758

4

Vibrio river

ATCC1.1611

5

Vibrio ven...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection kit of vibrio parahaemolyticus and a detection method thereof and specifically relates to a group of primers for detecting the vibrio parahaemolyticus, 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 parahaemolyticus and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Vibrio parahaemolyticus (Vp for short) is a Gram-negative halophilic bacterium widely distributed in offshore areas, salt lakes, fish, shellfish and other seafood, and is an important pathogen causing food poisoning in coastal areas. It can cause typical gastroenteritis reactions such as diarrhea, intestinal cramps, nausea, vomiting, and fever in patients, and severe cases can cause sepsis. In recent years, food poisoning caused by Vibrio parahaemolyticus infection has become one of the serious foodborne public health problems worldwide. In Japan, food poisoning caused by this bacterium accounts for 20% to 30% of the national food poisoning cases; data from the National Foodborne Disease Surveillance Network in my country shows the occurrence scale of food poisoning caused by V...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/63
Inventor 曾静魏海燕张蕾张西萌张琳马丹
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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