CN102758020A

CN102758020A - Multiple polymerase chain reaction (PCR) method for detecting gum porphyrin monad pathogenicity island genotype rapidly

Info

Publication number: CN102758020A
Application number: CN201210275907XA
Authority: CN
Inventors: 肖水清; 王兆强; 刘毅; 张玉杰
Original assignee: 肖水清
Priority date: 2012-08-06
Filing date: 2012-08-06
Publication date: 2012-10-31

Abstract

The invention relates to a multiple PRC method for detecting four types of rag genotypes of an oral cavity common pathogenic bacterium gum porphyrin monad and belongs to the technical field of molecular biology. Specific genes of the four types of rag of the gum porphyrin monad in the oral cavity are increased through the multiple PRC and are detected by using electrophoresis. By the aid of the primer design of the four types of rag genotypes of gum porphyrin monad, the reaction conditions in the PCR rapid detection are consistent, the method is sensitive and the specificity is high, detection identification of the four types of rage genotypes in clinic specimen is performed, the distribution of different rag genotypes is determined, the detection speed is increased greatly, and the detection efficiency is improved greatly.

Description

The genotypic multiple PCR method of rapid detection porphyromonas gingivalis pathogenicity island

Technical field

The invention belongs to technical field of molecular biology, relate to a kind of multiple PCR method of common 4 kinds of genotype detection of oral cavity pathogenic bacteria porphyromonas gingivalis pathogenicity island Rag gene.

Background technology

Porphyromonas gingivalis (P.gingivalis; Pg) be the Gram-negative obligatory anaerobic bacteria; Can cause periodontal tissue to destroy by a large amount of virulence factors of secretion, closely related with chronic periodontitis, aggressive periodontitis, periodontal abscess and dental pulp infection and occurring together property general systemic disease.Since Curtis in 1999 etc. found Pg pathogenicity island (pathogenicity islands), people had had further understanding to Pg pathogenic, and pathogenicity island is present in the virulent strain, lacks in general low virulent strain or the avirulent strain.At present, to be determined be the pathogenicity island of P.gingivalis to the rag locus.Experiment confirm; The inactivation in rag site can reduce the pathogenic of porphyromonas gingivalis, reduces its destruction to periodontal tissue, and there are 4 kinds of different gene types in the rag site in clinical strains; Difference called after rag-1, rag-2, rag-3 and rag-4; The pathogenecity of different genotype is also different, and rag-1 type Pg is the strongest to the destructiveness of periodontal soft tissue, is high virulence gene type; For confirming the pathogenicity island genotype of Pg in the clinical strains as early as possible; Be necessary to set up fast, sensitive detecting method detects the rag genotype in the clinical samples simultaneously; Confirming its destructiveness, the change of illness state of the pathogenesis of research periodontopathy, monitoring periodontopathy, confirm regimen to periodontal tissue.At present existing bibliographical information detects the research report of porphyromonas gingivalis and pathogenicity island gene respectively with round pcr; But each reaction only detects a kind of rag genotype; Can not meet clinical needs, and set up the four kinds of genotypic multiplex PCRs of rag that increase simultaneously just can identify Pg through primary first-order equation four kinds of genotypic pathogenicity island genes.

Summary of the invention

The objective of the invention is provides a kind of multiple PCR technique to detect four kinds of rag genotype of porphyromonas gingivalis simultaneously to above-mentioned technical problem, only just can identify four kinds of different rag genotype through a pcr amplification, improves detection efficiency and reduces the detection cost.

Multiple PCR technique comprises 4 kinds of genotypic special primers of rag of design amplification Pg, as follows:

Figure 201210275907X100002DEST_PATH_IMAGE001

The multiple PCR fast detecting method of 4 kinds of rag genotype specific genes of porphyromonas gingivalis comprises the steps:

1) the used dna profiling of amplification is to extract with the heating pyrolyze method: with specimen centrifuge, abandon supernatant, in deposition, add lysis buffer, and 100 ℃ of water-bath 10 min, centrifugal again, get supernatant as template, in-20 ℃ of preservations;

Said lysis buffer is: 1.0mmol/L EDTA, 1.0% Triton X-100,10mmol/L Tris-HCl, pH8.0.

2) the original liquid of 4 pairs of primers of not diluted is mixed the back and dilute by the amount of 1 pair of primer dilution, the primer after the dilution is as application liquid, and meter is made pm, and the primer meter behind 4 kinds of genotypic primer mixed dilutings of rag is made pn;

3) electrophoresis detection is identified.

Foregoing multiple PCR fast detecting method, preferred scheme be, step 1) during with specimen centrifuge controlled variable be 12 000 r/min, 2 min, maximum centrifugal radius are 6 cm.

Foregoing multiple PCR fast detecting method, preferred scheme is step 2) reaction system:

10×TransStart?Taq?Buffer 2.5ul，

Primer mixture (pn) 2.0ul,

TransStart?Taq?DNA?Polymerase 0.5ul，

dNTPs 0.5ul，

Template DNA 5.0ul,

Aseptic double-distilled water 14.5ul,

TV is 25 μ l

Foregoing multiple PCR fast detecting method, preferred scheme is step 2) PCR reaction cycle parameter is: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 30sec, 33 circulations; Last 72 ℃ are extended 10min, get 10 μ l multi-PRC reaction products and carry out 1% agarose gel electrophoresis detection specific gene.

The genotypic specific gene of the different rag of porphyromonas gingivalis in the said multiplex PCR amplification oral cavity comprises the steps:

The used dna profiling that increases is to extract with the heating pyrolyze method, and concrete steps are following:

With the specimen centrifuge of collecting (12 000 r/min, 2 min, maximum centrifugal radius are 6 cm), abandon supernatant; In deposition, add lysis buffer, 100 ℃ of water-bath 10 min, centrifugal again (12 000 r/min; 2min), get supernatant as template, subsequent use in-20 ℃ of preservations.

Above working method and experiment reagent are this area routine operation and commercial reagent if no special instructions.

Description of drawings

The detected result figure of Fig. 1 for obtaining through detection technique of the present invention;

Wherein, 1) Marker mark; 2) rag-1 type; 3) rag-1 type; 4) rag-2 type; 5) rag-1 type+rag-4 type; 6) rag-3 type; 7) rag-4 type.

Embodiment

Below through instance the present invention is done further description.

Embodiment:

The present invention relates to 4 kinds of genotypic multiplex PCRs of rag of rapid detection porphyromonas gingivalis, this method is utilized 4 kinds of rag genotype of multiplex polymerase chain re-action (PCR) synchronous detection porphyromonas gingivalis, and step is following:

(1) collection of periodontal pocket and gingival sulcus sample: adopt aseptic paper point method collect specimen respectively in patient's periodontal pocket or the gingival sulcus, concrete acquisition method is following: after conventional clear water was gargled, normal saline flushing was removed swill; Cotton balls dries up at a distance from wet, and the aseptic paper point is inserted in the periodontal pocket or in the gingival sulcus; When resistance, stop to insert; Take out after stopping 30s, put into the EP pipe of 1ml PBS ,-20 ℃ of preservations are to be measured.

(2) preparation of template DNA:, abandon supernatant with specimen centrifuge (12 000 r/min, 2 min, maximum centrifugal radius are 6 cm); In deposition, add lysis buffer, 100 ℃ of water-bath 10 min, centrifugal again (12 000 r/min; 2min), get supernatant, in-20 ℃ of preservations as template.

(3) multi-PRC reaction:

Draw 14.5 μ l aseptic deionized waters and add in the micro-centrifuge tube, add 10 * TransStart Taq Buffer, 2.5 μ l again, dNTPs 0.5 μ l; TransStart Taq DNA Polymerase 0.5 μ l; Primer mixed solution 2.0 μ l, template DNA 5.0 μ l, the centrifugal mixing of low speed centrifuge; Put into the PCR appearance, carry out pcr amplification reaction: 94 ℃ of 5min according to following parameter; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 30sec, 33 circulations; 72 ℃ are extended 10min;

The primer is following:

The original liquid of 4 pairs of primers of not diluted is mixed the back dilute by the amount of 1 pair of primer dilution, the primer after the dilution is as application liquid, and meter is made pm, and the primer meter behind 4 kinds of genotypic primer mixed dilutings of rag is made pn.

(4) PCR product electrophoresis detection:

Prepare 2% sepharose solution with the TAE damping fluid, heating for dissolving, to be cooled during to 60 ℃ of left and right sides, add ethidium bromide; Making its final concentration is 1 μ g/ml, and fully mixing is poured in the glued membrane; Put comb well, treat that gelling is solid after, remove comb; Gel is put into electrophoresis chamber, add the TAE damping fluid, make liquid level exceed gel surface 1-2mm; Again the sample behind the PCR is mixed with sample-loading buffer, sample is added in the well successively, cover electrophoresis chamber and energising with micropipet; 90V constant voltage electrophoresis moves DNA anode direction, behind the about 30min of electrophoresis; Cut off the electricity supply; Take out gel, under UV-light, observe, atlas analysis is according to having or not porphyromonas gingivalis and the 4 kinds of genotypic multiple PCR products expection of rag amplified fragments thereof to judge whether to contain the said gene type.The genotypic multiple PCR products expection of 4 kinds of rag of porphyromonas gingivalis expanding fragment length is respectively 628bp, 979bp, 423bp and 739bp.

The detected result figure of Fig. 1 for obtaining through detection technique of the present invention, figure can find out thus, from Lane2 to Lane7, is respectively the detected result of 6 parts of clinical samples, wherein, and Lane2 and 3, the amplified band size is 628bp, all is the rag-1 type; Lane4 is rag-2, and the DNA size is 979bp; Lane5 is rag-1 and rag-4 mixed type, comprises two bands that differ in size of 628bp and 739bp; Lane6 is the rag-3 type, and the DNA size is 423bp; Lane7 is the rag-4 type, and the DNA size is 739bp.

Claims

1. the genotypic multiple PCR method of rapid detection porphyromonas gingivalis pathogenicity island is characterized in that, adopts multiplex PCR to detect four kinds of pathogenicity island genotype: rag-1, rag-2, rag-3 and rag-4 of porphyromonas gingivalis simultaneously.

2. multiple PCR method according to claim 1 is characterized in that, as follows:

1) extract the used dna profiling of amplification with the heating pyrolyze method: with specimen centrifuge, abandon supernatant, in deposition, add lysis buffer, 100 ℃ of water-bath 10 min, centrifugal again, get supernatant as template, in-20 ℃ of preservations;

2) the primer is following:

Figure 201210275907X100001DEST_PATH_IMAGE001

The original liquid of 4 pairs of primers of not diluted is mixed the back dilute by the amount of 1 pair of primer dilution, the primer after the dilution is as application liquid, and meter is made pm, and the primer meter behind 4 kinds of genotypic primer mixed dilutings of rag is made pn;

3) electrophoresis detection is identified.

3. multiple PCR method as claimed in claim 2 is characterized in that, the used dna profiling that increases is to extract with the heating pyrolyze method, and concrete steps are following: with specimen centrifuge (12 000 r/min that collect; 2 min, maximum centrifugal radius are 6 cm), abandon supernatant; In deposition, add lysis buffer, 100 ℃ of water-bath 10 min, centrifugal again (12 000 r/min; 2min), get supernatant as template, subsequent use in-20 ℃ of preservations.

4. multiple PCR method as claimed in claim 2 is characterized in that, presses following reaction system:

10×TransStart?Taq?Buffer 2.5μl，

Primer mixed solution pn 2.0 μ l,

TransStart?Taq?DNA?Polymerase 0.5μl，

dNTPs 0.5μl，

Template DNA 5.0 μ l,

Aseptic double-distilled water 14.5 μ l;

TV is 25 μ l.

5. multiple PCR method as claimed in claim 2 is characterized in that, presses following reaction parameter: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 30sec, 33 circulations; Last 72 ℃ are extended 10min, get 10 μ l multi-PRC reaction products and carry out 1% agarose gel electrophoresis.