Campylobacter jejuni fluorescence quantitative PCR detection kit and detection method thereof

A technology for quantification of Campylobacter jejuni and fluorescence, which is applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., can solve the cost of false positives, reduce the detection target of operation steps, increase the possibility of contamination and detection The difficulty and other issues, to achieve the effect of strong specificity, improved sensitivity and specificity, and avoid missed and false detections

Inactive Publication Date: 2010-11-17
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Campylobacter jejuni is sublethal after being injured during food processing, and enters a "non-culturable state" in water, thus further increasing the possibility of contamination and the difficulty of detection
At present, the kit screening method for the detection of Campylobacter jejuni reported at home and abroad, although the operation steps are reduced and the detection target is narrowed down, there are still problems such as false positives and high cost.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The composition of embodiment 1 kit of the present invention

[0022] 1. The composition of the basic kit: (1) PCR MIX reaction solution, its components are 5×FQ PCR buffer, primer mixture with a concentration of 10 μM (the concentration of each primer is 10 μM), and a TaqMan probe mixture with a concentration of 10 μM (each The probe concentration is 10μM), the concentration of dNTPS and ddH is 10mM 2 O, the volume mixing ratio of each component is 10:1:0.5:1:30.5; the primer mixture contains: upstream primer 16S rRNA-F, downstream primer 16S rRNA-R, upstream primer hipO-F and downstream primer hipO-R , TaqMan probe mix includes: 16S rRNA-P and hipO-P; (2) Taq enzyme; (3) positive standard (1×10 7 copies / ml) and negative standard.

[0023] (1) Components in the PCR MIX reaction solution:

[0024] I: 5×FQ PCR buffer composition: 250mM Tris-HCl (pH8.0), 35mM MgCl 2 , 250 mM KCl and 10% (volume concentration) DMSO.

[0025] II: The design and preparation of primers a...

Embodiment 2

[0038] Embodiment 2 The method and verification thereof for the detection of Campylobacter jejuni by the kit of the present invention

[0039] Artificially add 1-10CFU, 10-100CFU bacteria into 225mL Bolton selective enrichment broth, under microaerophilic conditions, culture at 30°C for 3h, then at 37°C for 2h, and finally at 42±1°C for 19-43h, to obtain growth bacteria liquid.

[0040] (1) detect with kit of the present invention

[0041] Use the bacterial nucleic acid extraction kit to extract the DNA of the enrichment solution and the positive control strain. Take the Campylobacter jejuni PCR MIX reaction solution and Taq enzyme system prepared above, melt at room temperature, shake and mix well, and then centrifuge at 10,000rpm for 10s. According to each detection reaction system, add 24 μL PCR MIX and 2 μL Taq enzyme into an appropriate volume of clean PCR amplification tube or well, mix thoroughly and centrifuge, then add 4 μL DNA of the enrichment solution (add positiv...

Embodiment 3

[0045] Embodiment 3 detects the sensitivity of campylobacter jejuni with kit of the present invention

[0046] Dilute 0.1ml of original enrichment solution, 0.1ml of 100-1,000CFU gradient Campylobacter jejuni dilution, 0.1ml of 10-100CFU gradient of Campylobacter jejuni dilution, 0.1ml of 1-10CFU gradient of Campylobacter jejuni dilution DNA extraction solution was used to extract nucleic acid with bacterial nucleic acid extraction kit, and 4 μl of DNA extraction solution was used for fluorescent real-time PCR reaction. The results confirmed that the Ct value of nucleic acid extracted from 0.1ml of the original enrichment solution was 10.3; the Ct value of nucleic acid extracted from 0.1ml of 100-1,000CFU gradient bacterial dilution was 32.9; the Ct value of nucleic acid extracted from 0.1ml of 10-100CFU gradient The Ct value of the nucleic acid was 36.9; the Ct value of the nucleic acid extracted from 0.1ml of bacterial dilutions with a gradient of 1 to 10 CFU was 39.8, while...

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Abstract

The invention provides a campylobacter jejuni fluorescence quantitative PCR detection method and a detection kit thereof. The method can rapidly and accurately detect the campylobacter jejuni and reduces the false positive. The kit comprises the following reagents: PCR MIX reaction liquid, 5*FQ PCR buffer, a primer mixture with the concentration of 10 mu M, a TaqMan probe mixture with the concentration of 10 mu M, and dNTPs and ddH2O with the concentration of 10 mM, wherein the primer mixture comprises a 16S rRNA upstream primer F, a 16S rRNA downstream primer R, and an upstream primer hipO-F and a downstream primer hipO-R; and the TaqMan probe mixture comprises: (1) 16S rRNA-P and hipO-P; (2) Taq enzyme; and (3) positive standard substances and negative standard substances. By introducing high-efficiency and strong-specificity amplification primers and fluorescent probes, the sensitivity and specificity of detecting the campylobacter jejuni are improved and missing detection and error detection are avoided.

Description

technical field [0001] The invention relates to a nucleic acid detection kit for Campylobacter jejuni and a detection method thereof, belonging to the field of biotechnology. Background technique [0002] Campylobacter jejuni (Campylobacter jejuni) is an important pathogenic bacterium that is widely prevalent in the world. It not only causes a variety of livestock and poultry diseases, but also causes food poisoning in humans. Studies have proved that people infected with Campylobacter jejuni, Most cases are directly or indirectly related to animal food. Among all kinds of bacterial food poisoning in various countries, food poisoning caused by Campylobacter jejuni is often in the forefront, so the World Health Organization has listed this disease as one of the most common foodborne diseases. [0003] Campylobacter jejuni can be found in many types of food, including raw meat, poultry, eggs, dairy products, fish, shrimp and other foods. Campylobacter jejuni is in a subletha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 蒋原祝长青薛峰栾军邵景东陈国强唐泰山张常印张睿沈崇钰吴斌
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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