CN102154501A - PCR (Polymerase Chain Reaction) detection kit and method for two soybean germs - Google Patents

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) detection kit and method for diaporthe phaseolorum var. meridionalis (DPM) and diaporthe phaseolorum var. caulivora (DPC). In the kit and the detection method, a pair of specific primers is designed and synthesized according to ITS (Internal Transcribed Spacer) region sequences of two plant pathogenic fungi DPM and DPC respectively; the extracted DNA (Deoxyribonucleic Acid) is amplified by using a PCR technology, so that PCR segments of 270bp and 339bp are amplified respectively; a positive quality control is developed; and thus, the two fungi can be qualitatively detected, convenience, high speed, high specificity and high sensitivity are achieved, the DPM and the DPC carried by imported and exported soybeans can be detected and DPM and DPC epidemic situations of soybean planting areas at home and abroad can be monitored, the epidemic situations can be timely prevented and controlled, and the kit and the detection method have great popularization values and practical values.

Description

PCR detection kit and the detection method of two kinds of soybean germs

Technical field

The present invention relates to a kind of detection kit and detection method, particularly a kind of soybean south stem canker germ (DPM) and soybean north stem canker germ (DPC) PCR detection kit and detection method.

Background technology

Soybean south stem canker germ ( Diaporthe phaseolorum(Cke. ﹠amp; Ell.) Sacc.var. MeridionalesMorgan-Jones is called for short DPM) and soybean north stem canker germ ( Diaporthe phaseolorum(Cke. ﹠amp; Ell.) var. caulivora Athow ﹠amp; Caldwell is called for short DPC) be the inward Plant Quarantine harmful organism of China.Because that these two kinds of diseases take place in recent years abroad is general, economic impact is big, it is high and do not have in China and to report to import risk into, is decided to be 7 kinds of dangerous fungal diseases on the soybean by domestic relevant expert.It is big that DPM, DPC in soybean producing countries such as the U.S., Brazil, Argentina area take place, the production loss Ceng Gaoda 100% that causes.China is the big country of plantation and imported soybean, annual all the harvesting and a large amount of soybean of import.Soybean distributes very wide in China, cultivated area accounts for 8-10% of grain cultivated area throughout the year, is only second to paddy rice, wheat, corn, occupies critical role in national economy.And in China's imported soybean more than 95% from the U.S., Brazil, Argentina and Uruguay, China's imported soybean total amount reached more than 5,400 ten thousand tons unexpectedly in 2010, interdependency also from 2000 years 48.1% increase to 2007 years 78.7%.

Because traditional morphology qualification time is long, operation steps complexity, loaded down with trivial details, only the pure culture time behind the isolated strains just reaches 40-60 days, fast, is accurately diagnosed these two kinds of germs to bring difficulty.In recent years, the diagnostic method that generally adopts at these two kinds of fungies was morphological feature, PCR-RFLP, fluorescence PCR detecting method and order-checking, but clearly, PCR-RFLP, fluorescent PCR detection spend all very high.

Summary of the invention

Technical problem to be solved by this invention is at the deficiencies in the prior art, provide a kind of and can realize fast, accurately, cost is low, soybean south stem canker germ (DPM) easy to utilize and northern stem canker germ (DPC) PCR of soybean detection kit.

Another technical problem to be solved by this invention provides the method that detects with above-described detection kit.

Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of soybean south stem canker germ and soybean north stem canker germ PCR detection kit, is characterized in that it is made of following article:

1), negative control, composition is 1 of a 1mL sterilization distilled water;

2), positive control, the reagent composition is 1 of the DPM bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

3), positive control, the reagent composition is 1 of the DPC bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

4), dNTP is 1;

5), do not contain Mg ²⁺1 of PCR damping fluid;

6), contain Mg ²⁺1 of damping fluid;

7), primer is to DPMF ₃/ DPMR ₂

DPMF ₃For: 1 of 5 '-CCGAAACTCTGAGCAAAAAACAC-3 ';

DPMR ₂For: 1 of 5 '-CCTGGCGAGCCCGCCACTAG-3 ';

8), primer is to DPCF ₄/ DPCR ₂

DPCF ₄For: 1 of 5 '-GCCCCCTTGGGGGCCCCCC-3 ';

DPCR ₂For: 1 of 5 '-TCCTGGCGAGCTCGCCAATGA-3 ';

9), archaeal dna polymerase is 1;

10), the sterilization distilled water is 1;

11), the packing box, a cystose, its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that is no less than above-mentioned tubule quantity is arranged on the cystose, and above-mentioned each tubule correspondence respectively is positioned in these apertures;

Positive control is deposited separately; Test kit is in-20 ℃ of preservations.

Technical problem to be solved by this invention can also further realize by following technical scheme.The present invention also provides the PCR detection method of a kind of soybean south stem canker germ (DPM), soybean north stem canker germ (DPC), is characterized in: use the described test kit of above technical scheme; Wherein a kind of germ of each detection; During detection, only right with the Auele Specific Primer of a kind of germ wherein; Prepare following reagent before detecting: DNA extraction CTAB lysate, chloroform, primary isoamyl alcohol, Virahol, 70% ethanol, ultrapure water or TE solution; The detection step is as follows:

(1) extraction of dna profiling: the phytopathy tissue sample that 5-10 is restrained bacterial strain or be milled into powder is put into the 1.5mL centrifuge tube, adds 600 μ l CTAB lysates in 65 ℃ of following water-bath 30min; Taking out the back isopyknic volume ratio of adding is the chloroform of 24:1: primary isoamyl alcohol, and after the top is even, the centrifugal 10min of 12000-15000 r/min; Get and add the chloroform that isopyknic volume ratio is 24:1 behind the supernatant again: primary isoamyl alcohol, after the top is even, the centrifugal again 10min of 12000-15000r/min; Get supernatant add isopyknic aqueous isopropanol top even after, the centrifugal 10min of 12000-15000r/min; Abandon supernatant, get precipitation, add 70% ethanolic soln of 400 μ l after, the centrifugal 5min of 12000-15000r/min; Remove supernatant, natural air drying adds ultrapure water or the TE solution of 50 μ l, dna profiling, preserve standby down for-20 ℃;

(2) preparation of PCR premix, adopt following method respectively:

1. the increase preparation of PCR premix of DPM:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺ PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPMF ₃/ DPMR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

_____________________________________________

--cumulative volume: 50 μ l

2. the increase preparation of PCR premix of DPC:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺ PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPCF ₄/ DPCR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

__________________________________________

--cumulative volume: 50 μ l

(3) with step (2) 1. in the PCR premix PCR reaction tubes of packing into of amplification DPM, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×4min

94℃×30sec

60℃×30sec

35?cycles

72℃×30sec

72℃×10min

25 ℃ * 1min or end

With step (2) 2. in the PCR premix PCR reaction tubes of packing into of amplification DPC, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×5min

94℃×20sec

60℃×20sec

?35?cycles

72℃×20sec

72℃×10min

25 ℃ * 1min or end

(4) get the 10 μ l reaction solutions that add behind the 3 μ l tetrabromophenol sulfonphthalein mixings after amplified reaction finishes, through 2% agarose gel electrophoresis, voltage is by observing down in uv analyzer behind the length 5V/cm electrophoresis 40-50min, if band occurs at 270bp or 339bp place, then be respectively the DPM or the DPC positive, illustrate and carry DPM or DPC in positive control or the testing sample, otherwise negative, illustrate and do not carry DPM or DPC in negative control or the testing sample.

In the inventive method, when detecting DPM, add DPMF ₃/ DPMR ₂Primer does not add DPCF ₄/ DPCR ₂Primer, other reagent are constant.

The present invention designs respectively according to two kinds of fungi ITS regional sequences and has synthesized a pair of Auele Specific Primer, is respectively:

DPMF ₃:?5’-CCGAAACTCTGAGCAAAAAACAC-3’；

DPMR ₂:?5’-CCTGGCGAGCCCGCCACTAG-3’；

Amplification DPM fungi ITS zone, fragment length is 270bp, DPMF ₃/ DPMR ₂The sequence 1 that primer is seen sequence table to the concrete sequence of the target DNA (purpose fragment) of amplification.

DPCF ₄:?5’-GCCCCCTTGGGGGCCCCCC-3’；

DPCR ₂?:?5’-TCCTGGCGAGCTCGCCAATGA-3’；

Amplification DPC fungi ITS zone, fragment length is 339bp.DPCF ₄/ DPCR ₂The sequence 2 that primer is seen sequence table to the concrete sequence of the target DNA (purpose fragment) of amplification.

The present invention uses round pcr the purpose fungal DNA that extracts is increased, and amplifies 270bp and 339bp fragment respectively.Developed test kit on this basis, and set up the method for quick of regular-PCR to DPM, DPC, this method begins whole process from sample preparation and can be controlled within 6 hours, can be applicable to import and export DPM in the soybean, DPC detection and domestic and international soybean planting district DPC, DPM epidemic monitoring.Highly sensitive, the high specificity of test kit of the present invention and detection method thereof, accurately, reliable, fast, simple, easy to operate, be convenient to promote.Can replace conventional morphological feature, PCR-RFLP authentication method or the checking that complements each other with it.

Compare with existing PCR-RFLP, fluorescent PCR technology, when the present invention detects DPM or DPC, only add DPMF ₃/ DPMR ₂Primer to or DPCF ₄/ DPCR ₂Primer is to being to have or not DPM or DPC in the decidable sample; Do not need to add and digest and to judge with restriction endonuclease again after fluorescent probe or reaction finish; Therefore expense is far below fluorescent PCR and PCR-RFLP, and also shorten greatly than PCR-RFLP method detection time.

Description of drawings

Fig. 1 carries out gel electrophoresis figure behind the pcr amplification for adopting primer DPMF3/ DPMR2 to DPM; Among the figure: swimming lane 1, DPM bacterial strain positive control; Swimming lane 2-3, the soybean stem stalk of inoculation DPM bacterial strain; Swimming lane 4, the soybean seeds of inoculation DPM bacterial strain; Swimming lane 5, the soybean stem stalk of no DPM bacterium; Swimming lane 6, the sterilization distilled water (negative control) of no positive bacteria.

Fig. 2 carries out gel electrophoresis figure behind the pcr amplification for adopting primer DPCF4/ DPCR2 to DPM; Among the figure: swimming lane 1, DPC bacterial strain positive control; Swimming lane 2-3, the soybean stem stalk of inoculation DPC bacterial strain; Swimming lane 4-5, the soybean seeds of inoculation DPC bacterial strain; Swimming lane 6, the soybean seeds of no DPC bacterium; Swimming lane 7, the sterilization distilled water (negative control) of no positive bacteria.

Embodiment

Following with reference to accompanying drawing, further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not constitute restriction to its right.

Embodiment 1.A kind of soybean south stem canker germ and soybean north stem canker germ PCR detection kit, it is made of following article:

1), negative control, composition is 1 of a 1mL sterilization distilled water;

2), positive control, the reagent composition is 1 of the DPM bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

3), positive control, the reagent composition is 1 of the DPC bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

4), dNTP is 1;

5), do not contain Mg ²⁺1 of PCR damping fluid;

6), contain Mg ²⁺1 of damping fluid;

7), primer is to DPMF ₃/ DPMR ₂

DPMF ₃For: 1 of 5 '-CCGAAACTCTGAGCAAAAAACAC-3 ';

DPMR ₂For: 1 of 5 '-CCTGGCGAGCCCGCCACTAG-3 ';

8), primer is to DPCF ₄/ DPCR ₂

DPCF ₄For: 1 of 5 '-GCCCCCTTGGGGGCCCCCC-3 ';

DPCR ₂For: 1 of 5 '-TCCTGGCGAGCTCGCCAATGA-3 ';

9), archaeal dna polymerase is 1;

10), the sterilization distilled water is 1;

11), the packing box, a cystose, its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that is no less than above-mentioned tubule quantity is arranged on the cystose, and above-mentioned each tubule correspondence respectively is positioned in these apertures;

Positive control is deposited separately; Test kit is in-20 ℃ of preservations.

Embodiment 2.Utilize embodiment 1 described test kit to carry out the PCR detection method of soybean south stem canker germ (DPM), soybean north stem canker germ (DPC).Wherein a kind of germ of each detection; During detection, only right with the Auele Specific Primer of a kind of germ wherein; Prepare following reagent before detecting: DNA extraction CTAB lysate, chloroform, primary isoamyl alcohol, Virahol, 70% ethanol, ultrapure water or TE solution; The detection step is as follows:

(1) extraction of dna profiling: the phytopathy tissue sample that 5-10 is restrained bacterial strain or be milled into powder is put into the 1.5mL centrifuge tube, adds 600 μ l CTAB lysates in 65 ℃ of following water-bath 30min; Taking out the back isopyknic volume ratio of adding is the chloroform of 24:1: primary isoamyl alcohol, and after the top is even, the centrifugal 10min of 12000-15000 r/min; Get and add the chloroform that isopyknic volume ratio is 24:1 behind the supernatant again: primary isoamyl alcohol, after the top is even, the centrifugal again 10min of 12000-15000r/min; Get supernatant add isopyknic aqueous isopropanol top even after, the centrifugal 10min of 12000-15000r/min; Abandon supernatant, get precipitation, add 70% ethanolic soln of 400 μ l after, the centrifugal 5min of 12000-15000r/min; Remove supernatant, natural air drying adds ultrapure water or the TE solution of 50 μ l, dna profiling, preserve standby down for-20 ℃;

(2) preparation of PCR premix, adopt following method respectively:

1. the increase preparation of PCR premix of DPM:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺ PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPMF ₃/ DPMR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

_________________________________________

--cumulative volume: 50 μ l

2. the increase preparation of PCR premix of DPC:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺ PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPCF ₄/ DPCR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

____________________________________________

--cumulative volume: 50 μ l

(3) with step (2) 1. in the PCR premix PCR reaction tubes of packing into of amplification DPM, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×4min

94℃×30sec

60℃×30sec

35?cycles

72℃×30sec

72℃×10min

25 ℃ * 1min or end

With step (2) 2. in the PCR premix PCR reaction tubes of packing into of amplification DPC, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×5min

94℃×20sec

60℃×20sec

35?cycles

72℃×20sec

72℃×10min

25 ℃ * 1min or end

(4) get the 10 μ l reaction solutions that add behind the 3 μ l tetrabromophenol sulfonphthalein mixings after amplified reaction finishes, through 2% agarose gel electrophoresis, voltage is by observing down in uv analyzer behind the length 5V/cm electrophoresis 40-50min, if band occurs at 270bp or 339bp place, then be respectively the DPM or the DPC positive, illustrate and carry DPM or DPC in positive control or the testing sample, otherwise negative, illustrate and do not carry DPM or DPC in negative control or the testing sample.

Embodiment 3.Soybean south stem canker germ (DPM) and soybean north stem canker germ (DPC) PCR detection kit and detection method application experiment.

Experiment content: the primer, experiment material and the experimental technique that adopt Lianyun Harbour Entry-Exit Inspection and Quarantine Bureau to provide, detecting in the his-and-hers watches for test agent.

The test experiments material sees Table 1, and test sees Table 2 with primer.

Table 1. is for the examination list of materials

Sequence number	Title material
			1	Soybean stem stalk, the soybean seeds of inoculation DPM bacterium
2	Soybean stem stalk, the soybean seeds of inoculation DPC bacterium
		3	Dna sample: DPM, DPC
4	The soybean stem stalk or the soybean seeds of no positive bacteria
		5	The sterilization distilled water (negative control) of no positive bacteria

Table 2. test is tabulated with primer

Experimental technique: adopt embodiment 1 described test kit, test kit is provided by Lianyun Harbour Entry-Exit Inspection and Quarantine Bureau; Adopt embodiment 2 described methods to detecting for test agent.

Experimental result and conclusion: experiment detects sample, in the reaction system of using, negative control, positive control, positive, negative sample have been set up, positive control and known positive all amplify the dna fragmentation of corresponding size as a result, negative control and known negative sample all do not amplify nonspecific dna fragmentation, referring to Fig. 1, Fig. 2.Experiment described test kit of demonstration and the detection method feasibility of being set up are strong, the specificity height.

?

SEQ?ID?NO.1

＜110〉Lianyungang Entrance ﹠. Exist Inspection and Quarantine Administration, People's

＜120〉PCR detection kit and the detection method of two kinds of soybean germs

<160> 2

<170> PatentIn?version?3.3

<210> 1

<211> 270

<212> DNA

＜213〉amplification DPM fungi ITS regional gene segment

<400> 1

ccgaaactct?gagcaaaaaa?cacaaatgaa?tcaaaacttt?caacaacgga?tctcttggtt 60

ctggcatcga?tgaagaacgc?agcgaaatgc?gataagtaat?gtgaattgca?gaattcagtg 120

aatcatcgaa?tctttgaacg?cacattgcgc?cctctggtat?tccggagggc?atgcctgttc 180

gagcgtcatt?tcaaccctca?agcctggctt?ggtgttgggg?cactgcctgt?agaagggcag 240

gccctgaaat?ctagtggcgg?gctcgccagg 270

<210> 2

<211> 339

<212> DNA

＜213〉amplification DPC fungi ITS regional gene segment

<400> 2

gcccccttgg?gggccccccg?gagacgggga?gcagcccgcc?ggcggccaag?ctaactcttg 60

tttttacact?gaaactctga?gaaataaaca?taaatgaatc?aaaactttca?acaacggatc 120

tcttggttct?ggcatcgatg?aagaacgcag?cgaaatgcga?taagtaatgt?gaattgcaga 180

attcggtgaa?tcatcgaatc?tttgaacgca?cattgcgccc?tctggtattc?cggagggcat 240

gcctgttcga?gcgtcatttc?aaccctcaag?cctggcttgg?tgttggggca?ctgcctgtaa 300

aagggcaggc?cctgaaattc?attggcgagc?tcgccagga 339

Claims (2)

1. soybean south stem canker germ and soybean north stem canker germ PCR detection kit is characterized in that it is made of following article:

1), negative control, composition is 1 of a 1mL sterilization distilled water;

2), positive control, the reagent composition is 1 of the DPM bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

3), positive control, the reagent composition is 1 of the DPC bacterial strain DNA that is dissolved in 100ng in the 1mL sterilization distilled water;

4), dNTP is 1;

5), do not contain Mg ²⁺1 of PCR damping fluid;

6), contain Mg ²⁺1 of damping fluid;

7), primer is to DPMF ₃/ DPMR ₂

DPMF ₃For: 1 of 5 '-CCGAAACTCTGAGCAAAAAACAC-3 ';

DPMR ₂For: 1 of 5 '-CCTGGCGAGCCCGCCACTAG-3 ';

8), primer is to DPCF ₄/ DPCR ₂

DPCF ₄For: 1 of 5 '-GCCCCCTTGGGGGCCCCCC-3 ';

DPCR ₂For: 1 of 5 '-TCCTGGCGAGCTCGCCAATGA-3 ';

9), archaeal dna polymerase is 1;

10), the sterilization distilled water is 1;

11), the packing box, a cystose, its size is identical with the bottom surface of packing box, is loaded in the box; The aperture that is no less than above-mentioned tubule quantity is arranged on the cystose, and above-mentioned each tubule correspondence respectively is positioned in these apertures;

Positive control is deposited separately; Test kit is in-20 ℃ of preservations.

2. the PCR detection method of soybean south stem canker germ (DPM), soybean north stem canker germ (DPC), it is characterized in that: application rights requires 1 described test kit; Wherein a kind of germ of each detection; During detection, only right with the Auele Specific Primer of a kind of germ wherein; Prepare following reagent before detecting: DNA extraction CTAB lysate, chloroform, primary isoamyl alcohol, Virahol, 70% ethanol, ultrapure water or TE solution; The detection step is as follows:

(1) extraction of dna profiling: the phytopathy tissue sample that 5-10 is restrained bacterial strain or be milled into powder is put into the 1.5mL centrifuge tube, adds 600 μ l CTAB lysates in 65 ℃ of following water-bath 30min; Taking out the back isopyknic volume ratio of adding is the chloroform of 24:1: primary isoamyl alcohol, and after the top is even, the centrifugal 10min of 12000-15000 r/min; Get and add the chloroform that isopyknic volume ratio is 24:1 behind the supernatant again: primary isoamyl alcohol, after the top is even, the centrifugal again 10min of 12000-15000r/min; Get supernatant add isopyknic aqueous isopropanol top even after, the centrifugal 10min of 12000-15000r/min; Abandon supernatant, get precipitation, add 70% ethanolic soln of 400 μ l after, the centrifugal 5min of 12000-15000r/min; Remove supernatant, natural air drying adds ultrapure water or the TE solution of 50 μ l, dna profiling, preserve standby down for-20 ℃;

(2) preparation of PCR premix, adopt following method respectively:

1. the increase preparation of PCR premix of DPM:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPMF ₃/ DPMR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

___________________________________________

--cumulative volume: 50 μ l

2. the increase preparation of PCR premix of DPC:

To need with reagent after the centrifugal several seconds in the test kit before using, add various compositions by following art formula, each reagent except that dna profiling add in advance mix after, add dna profiling, positive control or the negative control that extracts at last, the zone that adds dna profiling separates with the zone that adds other reagent, and cumulative volume is 50 μ l;

The PCR premix adds the art formula:

dNTP　 4μl

Do not contain Mg ²⁺PCR damping fluid 5 μ l

Contain Mg ²⁺Damping fluid 5 μ l

Primer is to DPCF ₄/ DPCR ₂4 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization distilled water 26.5-29.5 μ l

Dna profiling or positive control or negative control 2-5 μ l

_______________________________________________

--cumulative volume: 50 μ l

(3) with step (2) 1. in the PCR premix PCR reaction tubes of packing into of amplification DPM, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×4min

94℃×30sec

60℃×30sec ?35?cycles

72℃×30sec

72℃×10min

25 ℃ * 1min or end

With step (2) 2. in the PCR premix PCR reaction tubes of packing into of amplification DPC, place gene-amplificative instrament, do not have the heat lid as instrument, add the capping of 1-2 dropstone wax oil; It is as follows that the PCR loop parameter is set, and increases:

94℃×5min

94℃×20sec

60℃×20sec

?35?cycles

72℃×20sec

72℃×10min

25 ℃ * 1min or end

(4) get the 10 μ l reaction solutions that add behind the 3 μ l tetrabromophenol sulfonphthalein mixings after amplified reaction finishes, through 2% agarose gel electrophoresis, voltage is by observing down in uv analyzer behind the length 5V/cm electrophoresis 40-50min, if band occurs at 270bp or 339bp place, then be respectively the DPM or the DPC positive, illustrate and carry DPM or DPC in positive control or the testing sample, otherwise negative, illustrate and do not carry DPM or DPC in negative control or the testing sample.

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