CN1733936A

CN1733936A - Spiroplasma pathogenic microorganism PCR fast checking technique

Info

Publication number: CN1733936A
Application number: CN 200510041005
Authority: CN
Inventors: 王文; 丁正峰; 顾伟
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2005-07-13
Filing date: 2005-07-13
Publication date: 2006-02-15
Anticipated expiration: 2025-07-13
Also published as: CN100363502C

Abstract

Disclosed is a PCR fast detection technique for Spiroplasma pathogen microorganisms comprising the following steps: (1) extracting template DNA in the detected sample with Chelex-100 method, (2) proceeding polymerase chain reaction with the 16S rDNA specific primer of the Spiroplasma, (3) subjecting the PCR product to electrophoresis detection, wherein the existence of Spiroplasma can be determined by the emergence of destination electrophoresis strip. The destination electrophoresis strip is at the position of 271bp, it can product certain diagnosis within 2-4 hours, and can carry out further semi-quantitative determination.

Description

Spiroplasma pathogenic microorganism PCR fast checking technique

Technical field

The present invention relates to the molecular Biological Detection technical field of pathogenic micro-organism, especially a kind of Spiroplasma pathogenic microorganism PCR fast checking technique.

Background technology

The spiral shell substance is the pathogenic micro-organism of finding the seventies in 20th century that parasitizes plant and insect, in recent years we have found this pathogenic micro-organism in river crab and small lobsters body, Auele Specific Primer with spiral shell substance 16S rna gene amplifies the 271bp band in sick crab and sick shrimp body, through order-checking with in the GenBank comparison, confirm that they are spiral shell substance quasi-microorganism.

At present, in conventional polymerase chain reaction (PCR) testing process, adopt phynol method or test kit to carry out the DNA extraction of test sample usually, these methods are loaded down with trivial details, time-consuming, the sample size that phynol method needs is big, and the DNA loss is many, and the intravital blood that is not suitable for trace detection and animal detects; Test kit then cost is higher, and use range limitation, and common a kind of test kit can only carry out template extraction at the sample of one or two kind of different sources.In addition, the PCR of existing pathogenic micro-organism detects just can be qualitative, and can not be quantitative.

Summary of the invention

The objective of the invention is at the important microbe pathogenic agent in the aquaculture of latest find---spiral shell substance, set up a kind of simple and easy, quick, responsive molecular Biological Detection technology.The present invention also further provides Spiroplasma pathogenic microorganism PCR sxemiquantitative Fast Detection Technique.

Technical scheme of the present invention is:

B. extract the template DNA that detects in the sample with the Chelex-100 method;

C. the 16S rDNA Auele Specific Primer with the spiral shell substance carries out polymerase chain reaction (PCR);

D. amplified production is through electrophoresis detection, the target electrophoretic band occurs and can determine to detect and have the spiral shell substance to exist in the sample, and said target electrophoretic band is meant the 271bp place.

Optimized technical scheme of the present invention is, e in steps also after steps d: the former bulk concentration of sxemiquantitative spiral shell, specifically be that the brightness of said target electrophoretic band with the electrophoretic band of the dilution standard sample of different gradients is compared, obtain detecting the former bulk concentration of spiral shell of sample.

The pathogenic bacteria that said detection sample can be bed mud, separation and Culture among the present invention, host tissue (comprising muscle, viscera tissue, blood etc.).At different detection samples, before extracting template DNA, carry out corresponding pre-treatment, to reach washing and the dense purpose that contracts of combining, can further improve the detection effect.

Said electrophoresis detection is recommended agarose electrophoresis or polyacrylamide gel electrophoresis in the steps d of the present invention.

The invention has the beneficial effects as follows: at first, adopt simple and easy, responsive Chelex-100 DNA extraction method, its cost has only 1/10 of conventional extracting method.Secondly, the susceptibility of this technology is high, can extract extremely micro-blood from live body and measure, thereby realize the purpose that live body detects, and in addition, this technology can also detect pathogenic agent from the bed mud that the shrimp crab perches, and the minimum concentration that can detect pathogenic agent reaches 10 ^-8(bacterium in both every ml volumes), susceptibility is far longer than technology such as light microscopic and Electronic Speculum.Sensitivity of the present invention, convenient, quick, cost is low, can go out to clarify a diagnosis at 2-4 hour.This invention not only can apply to the rapid detection of the spiral shell substance disease in the aquaculture, can also apply to any quarantine and the detection relevant with spiral shell substance disease.

Description of drawings

Fig. 1 is polyacrylamide gel electrophoresis detection figure;

Fig. 2 is agarose electrophoresis detection figure.

Embodiment

Embodiment 1: the testing environment bed mud

In July, 2004, in the pond of Anhui Quanjiang one crab tremble disease outburst, gather the bed mud sample a little, take back detection.Concrete operations are as follows:

A) pre-treatment: get about two spoons mud sample to be measured and be dissolved in a few hours in the 50mL water, stir during this time for several times, thorough mixing → quantitative paper is filtered, remove mud sample residue → filtrate is placed the 50mL centrifuge tube, 12000rpm, 4 ℃, centrifugal 1 hour → the careful supernatant that removes, bottom liquid is moved in the 1.5mLEppendorf pipe → 4 ℃ of 8000rpm, centrifugal 1 hour → carefully remove supernatant liquid, stay the about 0.1mL of layer in pipe;

B) template extraction: (1) Xiang Guanzhong adds 5%Chelex-100 150-200 μ L, thermal agitation 10 seconds; (2) 56 ℃ of water-baths 20 minutes, during mixing for several times; (3) took out thermal agitation 10 seconds, 99 ℃ of water-baths 8 minutes (strict control); (4) take out thermal agitation after 10 seconds, 13000rpm, 4 ℃, centrifugal 5 minutes; (5) shift supernatant (dna profiling) to another clean Eppendorf pipe ,-20 ℃ of preservations, amplification as early as possible.

C) the 16S rDNA Auele Specific Primer with the spiral shell substance carries out polymerase chain reaction (PCR), and the reaction cumulative volume is made as 25 μ L, PCR mix ingredients (every pipe): deionized water 14.85 μ L; STR 10XBuffer 2.5 μ L (Mg2+, dNTP wait mixture, purchase the company in Promega); Two kinds of primer mixture 2.5 μ L (concentration is 2 μ mol/L), Taq DNA Polymerase (0.75u) 0.15 μ L, template 5 μ L.Circulation is provided with parameter: 96 ℃, and 2 minutes → 94 ℃, 1 minute; 65 ℃, 1 minute; 72 ℃, 1.5 minutes (30 circulations) → 72 ℃ 10 minutes;

D) polyacrylamide gel electrophoresis detects: have target stripe (271bp) to occur, as shown in Figure 1, wherein the 1st road is detected target stripe from the mud sample (big or small 271bp), the 2nd road is the band that amplifies in the spiral shell substance of laboratory pure culture, M, Marker, be followed successively by 350bp from top to bottom, 222bp, 179bp.Detect the pathogenic bacterium spiral shell substance that contains tremble disease in the mud sample; Consistent with the result of negative staining electron microscope.

Embodiment 2: the pathogenic bacteria that detects separation and Culture

Get the Spiroplasma pathogenic bacterium culture of 1mL laboratory pure culture, being diluted to the former bulk concentration of spiral shell is 10 ⁶Individual/mL, 13000rpm, centrifugal 30 minutes, removes supernatant by 4 ℃; Template extraction, PCR react with

embodiment

1,2% agarose electrophoresis shown in Fig. 2 the 6th road.M among Fig. 2, Marker is followed successively by 100bp from the bottom up, 200bp, 300bp, 400bp etc., spiral shell substance specific band appears between 200bp and the 300bp, is 271bp.

Detecting the former bulk concentration of spiral shell as stated above respectively is 10 ⁴Individual/mL, 10 ²The sample of individual/mL, the result is respectively shown in Fig. 2 the 7th road and the 8th road.

Embodiment 3: detect host's blood

During summer high-temperature in 2004, gather to suffer from the crab number of tremble disease,

A) pre-treatment: live body extracts the about 10 μ L of its blood, mixes in a small amount of PBS (pH=7.0), and mixing moves in the 1.5mL Eppendorf pipe.

B) template extraction: (1) adds 5%Chelex-100 150-200 μ L in the pipe that sample is housed, Proteinase K (final concentration is 100 μ g/mL) mixing; (2) 56 ℃ water-bath 1.5-2 hour, during mixing for several times; (3) took out thermal agitation 10 seconds, 99 ℃ of water-baths 8 minutes (strict control); (4) took out thermal agitation 10 seconds, 13000rpm, 4 ℃, centrifugal 5 minutes; (5) shift supernatant (dna profiling) to another clean Eppendorf pipe ,-20 ℃ of preservations, amplification as early as possible.

C) PCR reacts with embodiment 1,

D) 2% agarose electrophoresis detects: have target stripe to occur, shown in Fig. 2 the 1st road.

E) the former bulk concentration of sxemiquantitative spiral shell: as Fig. 1, the brightness in 1 road is between 6 roads and 7 roads, and the result shows that the former bulk concentration of spiral shell is about 10 ⁵Individual/mL.Consistent with the observations of light microscopic and Electronic Speculum.

Utilize widely used magnificent Shun on the present market to extract the test kit (W6501) of tissue, cell, blood DNA in a small amount, detect the blood of identical crab, shown in the 2nd road among Fig. 2, electrophoresis showed does not have target stripe to occur, the method of the susceptibility of this method not as this patent has been described, and limitation is obvious.

Embodiment 4: detect host's limb flesh

Get an ill crab appendage muscle a little, place 1.5mL Eppendorf pipe, shred with scissors as far as possible, template extraction, PCR reaction, electrophoresis detection are identical with embodiment 3, having detected target stripe occurs, shown in the 3rd road among Fig. 2, its spiral shell substance concentration result of sxemiquantitative shows its concentration of cause of disease about 10 ⁶Individual/mL, show that the spiral shell substance invaded muscle tissue, electron microscopic section also shows and contains more spiral shell substance in the muscle tissue.

Utilize magnificent Shun's test kit (W6501) detected spiral shell substance band from the limb flesh of identical crab, shown in the 4th road among Fig. 2, detectable level and Chelex-100 meet.

Embodiment 5: the negative control experiment

Detecting sample is tissue, the blood of healthy crab individuality, intestinal bacteria, and the isolating white corpuscle of human blood, detection method is substantially the same manner as Example 3, and target stripe does not all appear in the result, shown in the 5th swimming lane among Fig. 1.

Claims

1, a kind of Spiroplasma pathogenic microorganism PCR fast checking technique comprises the steps:

C. the 16S rDNA Auele Specific Primer with the spiral shell substance carries out the polymerase chain reaction;

2, according to the said Spiroplasma pathogenic microorganism PCR fast checking technique of claim 1, it is characterized in that, e in steps also after steps d: the former bulk concentration of sxemiquantitative spiral shell, soon said target electrophoretic band compares with the brightness of the electrophoretic band of the dilution standard sample of different gradients among the step e, obtains detecting the former bulk concentration of spiral shell of sample.

3, according to the said Spiroplasma pathogenic microorganism PCR fast checking technique of claim 2, it is characterized in that pathogenic bacteria or host tissue that wherein said detection sample is bed mud, separation and Culture.

4, according to the said Spiroplasma pathogenic microorganism PCR fast checking technique of one of claim 1-3, it is characterized in that, before step b, a in steps also: wash or spissated pre-treatment to detecting sample.

According to the said Spiroplasma pathogenic microorganism PCR fast checking technique of claim 4, it is characterized in that 5, said step a is:

Get mud sample to be measured dissolving and evenly filter with quantitative paper the back, filtrate is centrifugal, remove supernatant, bottom liquid is moved in the 1.5mL Eppendorf pipe centrifugal, remove supernatant liquid, stay the about 0.1mL of layer in pipe;

Or: live body extracts crab blood 10 μ L, mixes in a small amount of pH is 7.0 PBS, and mixing moves in the 1.5mL Eppendorf pipe;

Or: get crab appendage muscle a little, shred with scissors as far as possible, place 1.5mL Eppendorf pipe.