CN1944681A

CN1944681A - Method for quick detecting vibrio splendidus

Info

Publication number: CN1944681A
Application number: CN 200610134007
Authority: CN
Inventors: 王秀利; 常亚青; 田春雨; 仇雪梅; 马悦欣
Original assignee: Dalian Fisheries University
Current assignee: Dalian Fisheries University
Priority date: 2006-10-25
Filing date: 2006-10-25
Publication date: 2007-04-11

Abstract

The method of quick detecting Vibrio splendidus includes the following steps: 1. setting 25 microliter of PCR reaction reagent, upstream primer VsF of 5' -GGACGGGTGAGTAATGCCTAG-3' and downstream primer VsR of 5,-GTTAGCCGGTGCTTC TTCTG-3' in the PCR tube; 2. setting small amount of sample to be detected inside the PCR tube, performing amplification reaction with PCR instrument including the pre-denaturation at 94 deg.c for 4 min; the following 30 cycles of denaturation at 94 deg.c for 50 sec, annealing at 56 deg.c for 50 sec, extending at 72 deg.c for 50 sec; and final extending at 72 deg.c for 5 min and maintaining at 4 deg.c; and 3. mixing PCR product of the last step and PCR sampling buffer in 6 times, and performing DNA molecular weight marker detection in 1 % agarose gel electrophoresis with added ethidium bromide. The present invention has the advantages of high speed, high sensitivity, low cost, etc.

Description

The PCR method of rapid detection Vibrio splindidus

Technical field:

The present invention relates to the detection method of marine organisms pathogenic bacterium such as a kind of sea cucumber, especially a kind of PCR method of rapid detection Vibrio splindidus.

Background technology:

In recent years, the development of China mariculture industry rapidly, but the intensive culture pattern causes the sea farming disease serious day by day, breaks out to involve that scope is wide, mortality ratio is high, destructiveness is big, causes serious economy loss to national and raiser.Vibrios is the main conditioned pathogen of marine cultured animal that extensively distributes in seawater and marine organisms, wherein vibrio pathogen can make the death of aquaculture organism fulminant, will cause huge infringement to sea farming with virus, other bacteriums or parasite etc., be acknowledged as in the sea farming one of severe diseases the most, as to cause the pathogenic bacterium of the sea cucumber Beancurd sheet syndromes of juvenile sea cucumbers extensive dead (mortality ratio reaches 20%～50%) mainly be exactly Vibrio splindidus.Therefore, the detection for Vibrio splindidus is one of necessary means of preventing and treating disease.At present, a lot of for the detection method of germ, as microbiology, morphology, biological chemistry and PCR etc.PCR method wherein has the advantage that detection speed is fast, highly sensitive, cost is low.But, because PCR detection method be used to the to increase special primer of Vibrio splindidus not up to now, thus be methods such as microbial culture, morphology, biological chemistry for the detection of Vibrio splindidus so far, length consuming time (more than 2～3 days), cost height, detected result are very unreliable yet.

Summary of the invention:

The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, the PCR method of the rapid detection Vibrio splindidus that a kind of detection speed is fast, highly sensitive, cost is low to be provided.

Technical solution of the present invention is: a kind of method of rapid detection Vibrio splindidus is characterized in that having the following steps:

A. 25 μ lPCR reaction reagents are inserted in the PCR pipe, upstream, downstream primer in the reaction reagent are respectively:

VsF：5′-GGACGGGTGAGTAATGCCTAG-3′；

VsR：5′-GTTAGCCGGTGCTTCTTCTG-3′；

B. the detected sample of trace is put into the described PCR pipe of a step, used the PCR instrument and carry out amplified reaction, reaction conditions is: 94 ℃ of pre-sex change 4min, carry out following 30 circulations then: 94 ℃ of sex change 50s, 56 ℃ of annealing 50s, 72 ℃ are extended 50s; Last 72 ℃ are extended 5min, 4 ℃ of preservations.

C. the PCR product of getting 5 μ l b steps mixes with 6 * PCR sample-loading buffer, detects with the contrast of dna molecular amount mark in 1% agarose gel electrophoresis that adds ethidium bromide staining.

The component of described reaction reagent is as follows:

10 * PCR buffered soln, 2～3 μ l

4mmol/l?dNTP 2μl

VsF(25pmol/l) 1μl

VsR(25pmol/l) 1μl

Taq enzyme (5U/ μ l) 0.3 μ l

25mmol/l magnesium ion 1.5 μ l

The distilled water surplus.

The invention provides special primer VsF, the VsR of a pair of amplification Vibrio splindidus, thereby a kind of method of rapid detection Vibrio splindidus be provided, compare with prior art and have following advantage:

1. detection speed is fast, both without microbial culture also without extracting nucleic acid, can in 2～4 hours, obtain detected result;

2. highly sensitive, can be directly amplification and detect specific band from few bacterium, need not through pure culture and will reach just can detect after a certain amount of;

3. cost is low, owing to need not extracting nucleic acid, can directly it be merged to the PCR thermo-cracking, and the expense of having saved extracting nucleic acid has reduced the detection cost.

Embodiment:

A. 25 μ lPCR reaction reagents are inserted in the PCR pipe, the component of reaction reagent is as follows:

10 * PCR buffered soln, 2～3 μ l

4mmol/l?dNTP 2μl

VsF(25pmol/l) 1μl

VsR(25pmol/l) 1μl

Taq enzyme (5U/ μ l) 0.3 μ l

25mmol/l magnesium ion 1.5 μ l

The distilled water surplus.

C. the PCR product of getting 5 μ l b steps mixes with 6 * PCR sample-loading buffer, in 1% agarose gel electrophoresis that adds ethidium bromide staining, detect with dna molecular amount mark (DL2000) contrast, the nucleic acid fragment that has 405bp under ultraviolet lamp, then explanation has Vibrio splindidus.

Detect principle:

In the PCR stage of increasing in advance the sample thermo-cracking is discharged the DNA of pathogenic bacteria,, the Vibrio splindidus distinguished sequence is increased with the PCR instrument again with PCR reaction reagent and primer.Owing to the primer that the present invention is designed is peculiar by Vibrio splindidus, if therefore contain Vibrio splindidus in the sample, after passing through 1% agarose electrophoresis and ethidium bromide staining after the amplification, ultraviolet lamp detects the band that just can see a 405bp down so.

Claims

1. the PCR method of a rapid detection Vibrio splindidus is characterized in that having the following steps:

A. 25 μ l PCR reaction reagents are inserted in the 0.2ml PCR pipe, upstream, downstream primer in the reaction reagent are respectively:

VsF：5′-GGACGGGTGAGTAATGCCTAG-3′；

VsR：5′-GTTAGCCGGTGCTTCTTCTG-3′；

C. the PCR product of getting 5 μ l b steps mixes with 6 * PCR sample-loading buffer, detects in contrast with dna molecular amount mark in 1% agarose gel electrophoresis that adds ethidium bromide staining.

2. the PCR method of rapid detection Vibrio splindidus according to claim 1 is characterized in that the component of described reaction reagent is as follows:

10 * PCR buffered soln, 2～3 μ l

4mmol/l?dNTP 2μl

VsF(25pmol/l) 1μl

VsR(25pmol/l) 1μl

Taq enzyme (5U/ μ l) 0.3 μ l

25mmol/l magnesium ion 1.5 μ l

The distilled water surplus.