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LAMP (loop-mediated isothermal amplification) detection kit for Vibrio vulnificus and detection method using same

A technology of Vibrio vulnificus and detection method, which is applied in the biological field, can solve the problems of affecting PCR amplification results, difficulty in satisfying rapid identification, and high price, and achieve the effects of shortening reaction time, improving detection sensitivity, and short time consumption

Inactive Publication Date: 2013-06-19
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 for Vibrio vulnificus and detection method using same
  • LAMP (loop-mediated isothermal amplification) detection kit for Vibrio vulnificus and detection method using same
  • LAMP (loop-mediated isothermal amplification) detection kit for Vibrio vulnificus and detection method using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] Obtaining of monoclonal antibody against Vibrio vulnificus

[0045] The present invention can use the anti-Vibrio vulnificus monoclonal antibody that has been disclosed in the prior art. In order to achieve better detection effect, preferably, the present invention uses the wounded antibody secreted by the hybridoma cell line whose deposit number is CGMCC No.6755. Vibrio flagellin monoclonal antibody, the specific information and obtaining method of the monoclonal antibody have been described in detail in Chinese invention patent application 201210534092.2. 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 vulnificus (VV) (ATCC1758, stored in the laboratory) was inoculated in T1N1 medium (purchased from Beijing Land Bridge ...

Embodiment 2

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

[0104] (1) Immunomagnetic beads coupled with anti-Vibrio vulnificus monoclonal antibody; for example, the immunomagnetic beads prepared in Example 1; (2) LAMP reaction solution, which includes the outer upstream of the sequence table SEQ ID No.1 Primer (F3), the outer downstream primer (B3) shown in the sequence table SEQID No.2, the inner upstream primer (FIP) shown in the sequence table SEQ ID No.3, the inner downstream primer shown in the sequence table SEQ ID No.4 Primer (BIP), circular upstream primer (LF) shown in SEQ ID No.5 in the sequence table, circular downstream primer (LB) shown in SEQ ID No.6 in the sequence table; the primers are all entrusted to Dalian Bao Bioengineering Ltd Synthesis;

[0105] Further, the LAMP reaction solution also includes ThermoPol buffer, dNTPs, betaine and MgSO 4 ; Wherein ThermoPol buffer, purchased from NEB Company, 1 × ThermoPol buffer contains 0.1...

Embodiment 3

[0110] Embodiment 3: detect the specificity and sensitivity test of vibrio vulnificus kit

[0111] 1. Assay Specificity Analysis

[0112] The strains listed in Table 3 were detected by IMS-LAMP respectively, and the results showed that the amplified products of Vibrio vulnificus standard strains showed fluorescent green staining by SYBR GreenⅠ, and typical ladder-like amplification bands appeared by agarose gel electrophoresis; 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 vulnificus.

[0113] Table 3 list of experimental strains

[0114] serial number

strain name

serial number

1

Vibrio vulnificus

ATCC1758

2

Vibrio alginolyticus

ATCC1833

3

Vibrio parahaemolyticus

ATCC17802

4

Vibrio river

ATCC1.1611

5

Vibrio venis

ATCC1.1612

6

...

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Abstract

The invention discloses a LAMP (loop-mediated isothermal amplification) detection kit for Vibrio vulnificus and a detection method using the same, and particularly relates to a group of primers for detecting Vibrio vulnificus, a kit containing the primers and a detection method using the same. The primers have oligonucleotide sequences shown as SEQ ID NOs.1-6 in a sequence table. The kit disclosed by the invention is high in sensitivity and specificity, low in cost and simple to operate.

Description

technical field [0001] The invention relates to a LAMP detection kit for Vibrio vulnificus and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Vibrio vulnificus (VV) is a halophilic Vibrio that widely exists in seawater and seafood. The human body can be infected by raw seafood or contact with seawater and seafood through broken limb wounds. It is common in fishermen working at sea and sea drillers, causing sepsis and soft tissue infection. This bacteria is very harmful to patients with diabetes, alcoholic liver disease, liver cirrhosis, hepatitis, and liver dysfunction. It can cause Vibrio vulnificus sepsis, with rapid onset, rapid progression and high mortality. my country has listed the bacteria as the main inspection or monitoring object. At present, this bacterium is one of the pathogenic bacteria that must be inspected for most imported and exported aquatic products. [0003] At present, the method of detecting path...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
CPCY02A50/30
Inventor 曾静刘艳华张琳张蕾张西萌魏海燕
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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