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Fluorescent quantitative PCR method for detecting vibrio quantity in seawater

A fluorescent quantitative and vibrio technology, applied in the detection of Vibrio quantity in seawater, in the field of fluorescence quantitative PCR, can solve the problems of long cycle, large investment, inaccurate detection of Vibrio quantity, etc., achieving intuitive results and low operating environment requirements Effect

Inactive Publication Date: 2011-03-23
LIAONING OCEAN & FISHERIES SCI RES INST
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Problems solved by technology

[0004] The present invention solves the problems of inaccuracy, long period and large investment in the detection of the number of Vibrio in the traditional method, and adopts the fluorescent quantitative PCR method to detect the total number of Vibrio in the water body, and the specific content is as follows

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  • Fluorescent quantitative PCR method for detecting vibrio quantity in seawater

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Embodiment 3、2008

[0022] Example 3, August 2008, Wafangdian 40 acres of imitation japonicus culture ponds, take 1ml of pond water samples, 10-fold ratio dilution 3 gradients, each dilution 50μl respectively coated on TCBS plate and 2216E plate Above, each gradient was replicated twice, after standing for 30 minutes, and after inverting at 25°C for 24 hours, the plate counting method was used for counting. Some plates of TCBS medium were mold-growing and could not be counted. The dilution gradient of some plates was not enough and too dense to be counted. The total number of Vibrio bacteria obtained from counting effective plates was 8.4×10 7 cfu / mL, the total number of bacteria is 1×10 8 cfu / mL.

[0023] Take another 1L of pond water sample, filter it with a 0.8 μm microporous membrane, and filter the obtained filtrate with a 0.2 μm filter membrane, and keep the 0.2 μm pore size filter membrane. Cut the filter membrane into pieces and put it into a 5ml centrifuge tube, add 3ml sterile water a...

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Abstract

In the invention, vibrio quantity in aquaculture water is rapidly detected by fluorescent quantitative polymerase chain reaction (PCR) technology. A specific primer for vibrio bacteria is designed by taking a ribosome 16S rRNA gene as a target gene; a fluorescent quantitative standard curve is constructed in a mode of recombinant plasmid; a 20 mu L reaction system, which comprises 10 mu L of SYBR Green I fluorescent premixed reagent, 0.4 mu L of forward primer, 0.4 mu L of reverse primer, 1.6 mu L of DNA template and 7.6 mu L of deionized water, is established by a Premix Ex TaqTM reagent. The fluorescent quantitative PCR method has intuitive result, high specificity and high sensitivity, is rapid to detect the vibrio quantity, solves the problem of an inaccurate result caused by mold breeding in the conventional detection method, overcomes the defect of low specificity of a thiosulfate citrate bile salts sucrose agar (TCBS) culture medium for a vibrio quantitative detection method, and has long-term market development and economic investment potential.

Description

technical field [0001] The invention is a technique for detecting the quantity of Vibrio in seawater, which mainly adopts the fluorescent quantitative PCR method. This method makes up for the problems of TCBS medium easily polluted and low specificity in the traditional Vibrio quantity detection method, which lead to inaccurate test results, long test cycle and heavy workload, and can accurately, quickly and scientifically detect Vibrio in seawater. Bacteria can be quantified, which avoids the shortcomings of traditional methods, and belongs to the new field of biotechnology. Background technique [0002] Vibrio is one of the most common bacterial groups in the marine environment, and it is a normal dominant bacterial group in seawater, protozoa, fish and other marine organisms. Some species of Vibrio are pathogenic bacteria that can cause vibriosis in marine organisms, with aquatic products as intermediate hosts. Some Vibrio can cause diarrhea, bacteremia and trauma infect...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64C12R1/63
Inventor 汪笑宇周遵春关晓燕陈仲董颖姜北杨爱馥
Owner LIAONING OCEAN & FISHERIES SCI RES INST
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