CN1737161A - Human saliva S.mutans and S.sobrinus detection method - Google Patents
Human saliva S.mutans and S.sobrinus detection method Download PDFInfo
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- CN1737161A CN1737161A CN 200510019132 CN200510019132A CN1737161A CN 1737161 A CN1737161 A CN 1737161A CN 200510019132 CN200510019132 CN 200510019132 CN 200510019132 A CN200510019132 A CN 200510019132A CN 1737161 A CN1737161 A CN 1737161A
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Abstract
The invention discloses a human saliva S.mutans and S.sobrinus detection method, comprising the following steps: (1) designing primer, wherein the provided set type polymerase chain reaction includes double PCR and four pairs of primers, i.e. two pairs of forward primer and reverse primers of Streptococcus mutans and two pairs of forward primer and reverse primers of Streptococcus sobrinus, (2) extracting bacteria chromosome DNA and saliva chromosome DNA, (3) first PCR, charging the two pairs of primers, four kinds of dNTP mixture and template DNA and Taq enzyme, (4) second PCR, diluting the product of the first PCR, charging the other two pairs of primers for gene amplification, (5) carrying out electrophoresis. The invention realizes simple process and low cost.
Description
Technical field
The invention belongs to the oral epidemiology technical field, more specifically relate to the detection method of streptococcus mutans and streptococcus sobrinus in a kind of people's saliva.
Background technology
The dental caries disease is a kind of bacterial infection disease.Studies show that in a large number mainly causing the dental caries microorganism is the streptococcus mutans group.The streptococcus mutans group has several method.Generally be some independent bacterial classifications at present with the streptococcus mutans component.In the mankind generally popular serotype c and have the e of serum cross reaction with it and the f type be called " streptococcus mutans " (Streptococcus mutans, S.mutans).D, the g type is called streptococcus sobrinus or streptococcus sobrinus, and (Streptococcus sobrinus, S.sobrinus), preceding two kinds of bacteriums are higher in human recall rate.And other several streptococcus mutans group memberships, as the hamster suis (S.cricetus, serotype a), streptococcus muris-ratti (S.rattus, serotype b), monkey suis (S.macacae, serotype e), the road reins in suis (S.downei, serotype h), seldom in the mankind, detect.Therefore, streptococcus mutans and streptococcus sobrinus are human main pathogenic bacterium in the streptococcus mutans group.For a long time, mutans streptococcus receives much concern.But recent some studies show that streptococcus sobrinus is compared with streptococcus mutans, may have the stronger dental caries potentiality that cause, may be even more important to the generation and the activity of dental caries disease.When streptococcus mutans and streptococcus sobrinus exist simultaneously in saliva and the plaque, this only single streptococcus mutans or sick incidence of streptococcus sobrinus dental caries of existing significantly increases (Hirose H, Hirosek, Isogai E, eta1.Close association between Streptococcus sobrinus in the saliva of youngchildren and smooth-surface caries increment.Caries Res, 1993,27 (4): 292) (Lindquist B, Emilson CG, et al.Differences in cariogenicity betweenfresh isolates of Streptococcus sobrinus and Streptococcus mutans.CariesRes, 1991,25 (2): 146).The situation that streptococcus mutans infects separately is more common, and streptococcus sobrinus is everlasting and is existed on the tooth that maybe can isolate mutans streptococcus in host's mouth of more changeable chain.
The differentiation in the past and the method for streptococcus mutans and streptococcus sobrinus of identifying comprise: observe methods such as colonial morphology, biochemical identification, immunoserology evaluation, dna probe on light saliva substratum.But time-consuming, the not high shortcoming of sensitivity that these methods exist.
PCR is a kind of DNA amplification in vitro technology, have high specificity, highly sensitive, easy and simple to handle, save time, to characteristics such as starting materials specification of quality to be checked are low.
Recently, there are many investigators to detect oral cavity pathogeny bacterium with PCR.Allaker selects the middle 192bp fragment of mutans streptococcus surface protein gene (spaP gene) as the specific amplification fragment, detects dento enamel junction (enamel-dentine junction, the recall rate of streptococcus mutans EDJ) of decreasing at dental caries; Also the PCR method is detected the recall rate of streptococcus mutans and the Color of a kind of dental caries damage staining agent and done correlation analysis, the result shows that both are closely related, infers that fully removing painted tissue in the therapeutic process can prevent the remaining mutans streptococcus of possibility that the continuation of tissue of tooth is corroded.In addition, the scholar is arranged according to porphyromonas gingivalis (Porphyromonas gingivalis respectively, P.g) sequences Design Auele Specific Primers such as cilium gene (fimA gene), collagenase gene (collagenase gene), 16S rRNA gene in, carry out the PCR reaction, identifying porphyromonas gingivalis, and with the recall rate of porphyromonas gingivalis in PCR reaction detection different crowd plaque and the saliva.So far, with the rare both at home and abroad report of streptococcus mutans and streptococcus sobrinus in PCR reaction detection different crowd plaque and the saliva.
Summary of the invention
The detection method that the purpose of this invention is to provide streptococcus mutans and streptococcus sobrinus in a kind of people's saliva can rapid detection streptococcus mutans and streptococcus sobrinus, and method is simple, and is with low cost.
Three kinds of Transglucosylases of streptococcus mutans generation (glucosyltransferase, GTF): GTF-I (GTF of synthetic ISG), GTF-SI (GTF of synthesizing water-solubility and ISG), GTF-S (GTF of synthesizing water-solubility dextran).Streptococcus sobrinus produces four kinds of Transglucosylase-GTF-I, GTF-S, GTF-SA, GTF-SB.The GTF-I of mutans streptococcus gtfI gene and streptococcus sobrinus gtfB genes encoding can synthesize ISG, brings into play the important dental caries effect that causes.And in gtfB and the gtfI gene species specificity nucleotide sequence is arranged, therefore according to gtfB (M17361) (Shiroza T, Ueda S and KuramitsuK.Sequence analysis of the gtfB gene from Streptococcus mutans.JBacteriol, 1987,169 (9): 4263-4270) (Ueda S, Shiroza T, and Kuramitsu K.Sequenceanalysis of the gtfC gene from Streptococcus mutans GS-5.Gene, 1988,69 (1): 101-109) and gtfI (D90213) (Abo H, Matsumura T, Kodama T, et al.Peptidesequences for sucrose splitting and glucan binding within Streptococcus sobrinusglucosyltransferase (water-insoluble glucan synthetase) .J Bacteriol.1991,173 (3): nucleotide sequence 989-996) designs primer respectively, the primer of two kinds of bacteriums is mixed in the PCR reaction, can obviously differentiates two kinds of bacteriums.PCR can detect 〉=10 for the first time
5The CFU bacterium, PCR can detect 〉=10 for the second time
3The CFU bacterium.In order to achieve the above object, the present invention adopts following technical measures:
A: design primer.Nested polymerase chain reaction provided by the present invention (nested polymerasechain reaction, N-PCR) comprise secondary PCR, the 4 pairs of primers (streptococcus mutans first align anti-primer be 5 '-TAACTACACTTTCGGGTGGCTT and 5 '-GATGTATCAGTATAAGCGCCAG, second align anti-primer be 5 '-AAAGCAGATTCTAATGAATCGA and 5 '-AATGTAAAATTTTGCCATCAGC; Streptococcus sobrinus first align anti-primer be 5 '-GGTATCGTCCAAAATCAATCC and 5 '-TTATCGATACCGTAAGCTGCC, second align anti-primer be 5 '-TGGTATCGTCCAAAATCAATCC and 5 '-AGATTTGCAGTTGGTCAGCATC).
B: bacterial chromosomal dna and salivary chromosome DNA extraction.
The extraction of DNA of bacteria: 3ml bacterium culture overnight, 10,000rpm is centrifugal, 4 ℃ 10 minutes, remove supernatant, 200 μ lTE damping fluids flushings secondary, precipitation is resuspended in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃, 30 minutes, add 40 μ g Proteinase Ks, and adding 10%SDS45 μ l (final concentration 1.5%), mixing, 37 ℃, 3 hours, equal-volume phenol, chloroform extracting secondary, dehydrated alcohol deposit D NA, centrifugal 10,000rpm, 10 minutes, abandon supernatant, 70% washing with alcohol is dissolved in the TE damping fluid.
The extraction of salivary chromosome DNA: 1ml saliva, 8,000rpm, 4 ℃ are centrifugal, 15 minutes, remove supernatant, 500 μ lTE damping fluids are washed secondary, precipitation are resuspended in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃, 30 minutes, add 4 μ g Proteinase Ks, and add 10%SDS45 μ l (final concentration 1.5%) mixing, 37 ℃, 3 hours, equal-volume phenol, chloroform extracting secondary, dehydrated alcohol deposit D NA, centrifugal 1,0000rpm, 10 minutes, abandon supernatant, 70% washing with alcohol is dissolved in the TE damping fluid.
C: PCR for the first time.Add two couples of outer primer thp3, thp4, each 50pmol of thp5, thp6 successively, 4 kinds of dNTP mixtures (every kind of final concentration 200 μ M), 4 μ l, 10 * reaction buffer, 5 μ l, bacterium template DNA 2 μ l, TaqDNA polysaccharase 2.5U, aqua sterilisa 36 μ l, mixing.Reaction is by 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 30 seconds, and 58 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, circulates 30 times, last 72 ℃ of extensions 5 minutes.
D: PCR for the second time.For the first time the PCR product is got 2 μ l, dilute 10 times after, get 2 μ l and make template, with thp7, thp8, each 50pmol of thp9, thp10, other composition and step are with the PCR first time.
E: electrophoresis.Get amplified production 5 μ l, 1.0% agarose gel electrophoresis voltage 80V, the ingot dyeing of 0.5 μ g/ml bromination, with DL2,000DNA Marker is a standard control, observes amplified band under Ultraviolet Detector.
Compare with the bacterial cultivation of classics, N-PCR has the following advantages: 1. N-PCR can directly be template with the saliva, by PCR reaction, carries out the research of mutans streptococcus and streptococcus sobrinus recall rate in the oral cavity, and classical bacterial cultivation need be through separation and the evaluation of bacterium, complex steps; 2. N-PCR can identify from saliva fast and whether have streptococcus mutans and streptococcus sobrinus (only needing 6 hours), and classical bacterial cultivation relative complex generally needs 4-5 days; 3. N-PCR amplifies continuously through twice PCR and has improved the sensitivity and the specificity that detect, and classical bacterial cultivation identifies that according to ne ar and biochemical characteristic influenced by more human factor, sensitivity and specificity are low; 4. the result who carries out PCR at individual research object 1ml saliva sample can be divided into the infectation of bacteria level three level: 〉=105CFU, is the hyperinfection object; 〉=103-＜105CFU is the grade and moderate infection object;＜103CFU is for minuent infects object.Therefore; with the sleeve type PCR method when differentiating mutans streptococcus and streptococcus sobrinus; can be by comparing the difference on two kinds of bacterial numbers; suffer from situation (is index as losing benefit DMF with dental caries) in conjunction with dental caries; it is closer further to explore the sick susceptibility relation of which kind of bacterium and dental caries; be laying the foundation of pre-preventing decayed tooth disease, and classical bacterial cultivation there are not this characteristics.
Description of drawings
Fig. 1 is with thp3, and thp4, thp5, thp6 are primer pcr amplification mutans streptococcus group product electrophoresis synoptic diagram for the first time
1: hamster suis S.cricetus AHT; 2. streptococcus muris-ratti S.rattus BHT; 3. streptococcus mutans S.mutansIngbritt; 4. streptococcus sobrinus S.sobrinus OMZ176; 5. streptococcus mutans S.mutans LM-7; 6. streptococcus mutans S.mutans OMZ175; 7. streptococcus sobrinus S.sobrinus 6715; 8. suis S.downei Mfe28.Marker:DL2,000 Ladder. are reined in the road
Fig. 2 is with thp7, and thp8, thp9, thp10 are primer pcr amplification mutans streptococcus group product electrophoresis synoptic diagram for the second time
1: hamster suis S.cricetus AHT; 2. streptococcus muris-ratti S.rattus BHT; 3. streptococcus mutans S.mutansIngbritt; 4. streptococcus sobrinus S.sobrinus OMZ176; 5. streptococcus mutans S.mutans LM-7; 6. streptococcus mutans S.mutans OMZ175; 7. streptococcus sobrinus S.sobrinus 6715; 8. suis S.downei Mfe28.Marker:DL2,000 Ladder are reined in the road
Fig. 3 is with thp3, thp4, and thp5, thp6 are primer, different bacterium DNA is the PCR product electrophoresis synoptic diagram of template
1: streptococcus-salivarius S.salivarius HB; 2. streptococcus-salivarius S.salivarius HBC 12; 3. Streptococcus oralis S.oralis ATCC 10557; 4. light chain coccus S.mitis ATCC 9811; 5. Streptococcus sanguis S.sanguis903; 6. lattice Deng Shi suis S.gordonii; 7. actinomyces viscosus A.viscosus ATCC 19246; 8. actinomyces viscosus A.viscous ATCC 29525; 9. intestinal bacteria E.coli JM 109; 10. intestinal bacteria E.coliDH 5 α; 11. porphyromonas gingivalis P.g ATCC 33277; 12. pseudomonas Pseudomonas AP 930065; 13. streptococcus mutans S.mutans Ingbritt; 14. streptococcus sobrinus S.sobrinus 6715
Fig. 4 is with thp3, thp4, and thp5, thp6 are the sensitivity of the PCR of primer
Streptococcus mutans Ingbritt bacterium quantity is followed successively by: 1: 10
8CFU, 2: 10
7CFU; 3.10
6CFU; 4.10
5CFU
Fig. 5 is with thp3, thp4, and thp5, thp6 are the sensitivity of the PCR of primer
Streptococcus sobrinus 6715 bacterium quantity are followed successively by: 1:107CFU, 2:106CFU; 3:105CFU
Fig. 6 is with thp7, thp8, thp9, thp10 be primer the second time PCR sensitivity
Streptococcus mutans Ingbritt bacterium quantity is followed successively by: 1:104CFU, 2:103CFU
Fig. 7 is with thp7, thp8, thp9, thp10 be primer the second time PCR sensitivity
Fine hair shape suis S.sobrinus 6715 strain bacterium quantity are followed successively by: 1:105CFU, 2:104CFU;
3：103CFU
Fig. 8 is with thp3, thp4, and thp5, thp6 are primer, pcr amplification directly detects streptococcus mutans S.mutans and fine hair shape suis S.sobrinus. in the saliva
1. streptococcus mutans S.mutans Ingbritt; 2. fine hair shape suis S.sobrinus 6715; 3.A the saliva sample of research object; 4.B the saliva sample of object; 5.C the saliva sample of object
Fig. 9 is with thp3, thp4, and thp5, thp6 are primer, the secondary PCR amplification is streptococcus mutans S.mutans and fine hair shape suis S.sobrinus. in the detection saliva directly
1. streptococcus mutans S.mutans Ingbritt; 2. fine hair shape suis S.sobrinus 6715; 3.A the saliva sample of research object; 4.B the saliva sample of object; 5.C the saliva sample of object
Embodiment
With reference to the accompanying drawings the present invention is described in further detail below:
Three kinds of Transglucosylases of streptococcus mutans generation (glucosyltransferase, GTF): GTF-I, GTF-SI, GTF-S.Streptococcus sobrinus produces four kinds of Transglucosylase-GTF-I, GTF-S, GTF-SA, GTF-SB.The GTF-I of mutans streptococcus gtfI gene and streptococcus sobrinus gtfB genes encoding can synthesize ISG, brings into play the important dental caries effect that causes.And in gtfB and the gtfI gene species specificity nucleotide sequence is arranged, therefore the nucleotide sequence according to gtfB (M17361) and gtfl (D90213) designs primer (primer sequence sees Table 1) respectively, utilization shell type polymerase chain reaction,PCR technology uses the Taq archaeal dna polymerase to set up the PCR reaction system, amplifies the target DNA fragment (see Table 2 and Fig. 1-3) of different lengths from the mixt bacteria DNA that mutans streptococcus group type strain DNA of bacteria and saliva are extracted.
A: design primer.Nested polymerase chain reaction provided by the present invention (nested polymerasechain reaction, N-PCR) comprise secondary PCR, the 4 pairs of primers (streptococcus mutans first align anti-primer be 5 '-TAACTACACTTTCGGGTGGCTT and 5 '-GATGTATCAGTATAAGCGCCAG, second align anti-primer be 5 '-AAAGCAGATTCTAATGAATCGA and 5 '-AATGTAAAATTTTGCCATCAGC; Streptococcus sobrinus first align anti-primer be 5 '-GGTATCGTCCAAAATCAATCC and 5 '-TTATCGATACCGTAAGCTGCC, second align anti-primer be 5 '-TGGTATCGTCCAAAATCAATCC and 5 '-AGATTTGCAGTTGGTCAGCATC).
B: bacterial chromosomal dna and salivary chromosome DNA extraction.
The extraction of DNA of bacteria: 3ml bacterium culture overnight, 10,000rpm is centrifugal, 4 ℃ 10 minutes, remove supernatant, 200 μ lTE damping fluids flushings secondary, precipitation is resuspended in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃, 30 minutes, add 40 μ g Proteinase Ks, and adding 10%SDS45 μ l (final concentration 1.5%), mixing, 37 ℃, 3 hours, equal-volume phenol, chloroform extracting secondary, dehydrated alcohol deposit D NA, centrifugal 10,000rpm, 10 minutes, abandon supernatant, 70% washing with alcohol is dissolved in the TE damping fluid.
The extraction of salivary chromosome DNA: 1ml saliva, 8,000rpm, 4 ℃ are centrifugal, 15 minutes, remove supernatant, 500 μ lTE damping fluids are washed secondary, precipitation are resuspended in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃, 30 minutes, add 4 μ g Proteinase Ks, and add 10%SDS45 μ l (final concentration 1.5%) mixing, 37 ℃, 3 hours, equal-volume phenol, chloroform extracting secondary, dehydrated alcohol deposit D NA, centrifugal 1,0000rpm, 10 minutes, abandon supernatant, 70% washing with alcohol is dissolved in the TE damping fluid.
C: PCR for the first time.Add two couples of outer primer thp3, thp4, each 50pmol of thp5, thp6 successively, 4 kinds of dNTP mixtures (every kind of final concentration 200 μ M), 4 μ l, 10 * reaction buffer, 5 μ l, bacterium template DNA 2 μ l, TaqDNA polysaccharase 2.5U, aqua sterilisa 36 μ l, mixing.Reaction is by 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 30 seconds, and 58 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, circulates 30 times, last 72 ℃ of extensions 5 minutes.
D: PCR for the second time.For the first time the PCR product is got 2 μ l, dilute 10 times after, get 2 μ l and make template, with thp7, thp8, each 50pmol of thp9, thp10, other composition and step are with the PCR first time.
E: electrophoresis.Get amplified production 5 μ l, 1.0% agarose gel electrophoresis voltage 80V, the ingot dyeing of 0.5 μ g/ml bromination, with DL2,000DNA Marker is a standard control, observes amplified band under Ultraviolet Detector.
Table 1 streptococcus mutans and streptococcus sobrinus N-PCR primer sequence
| Bacterium | Primer | Sequence | The primer source | The position |
| S.mutans S.sobrinus S.mutans S.sobrinus | thp3 thp4 thp5 thp6 thp7 thp8 thp9 thp10 | 5′- | gtfB gtfI gtfB gtfI | 788-812 1295-1316 896-916 1575-1595 817-838 1264-1285 895-916 1537-1558 |
The product length of table 2 mutans streptococcus group amplification in the N-PCR reaction
| Bacterium | Bacterial strain and serotype | PCR product length (bp) | It is the same the same that source School of Stomatology oral cavity key lab of the biomedical engineering Ministry of Education of Wuhan University preserves strain | |
| PCR for the first time | PCR for the second time | |||
| Streptococcus (S.downei) is strangled in hamster streptococcus (S.cricetus) streptococcus muris-ratti (S.ratti) streptococcus mutans (S.mutans) streptococcus sobrinus (S.sobrinus) road | AHT(a) BHT(b) GS-5(c) ATCC10449(c) Ingbritt(c) LM-7(e) MT703(e) OMZ175(f) MT6219(f) OMZ176(d) 6715(g) OMZ65(g) Mfe28(h) | 0 0 529 700 | 0 0 468 663 | |
Through too much group electrophoresis evaluation, confirm streptococcus mutans and streptococcus sobrinus expanding fragment length (PCR 529bp and 700bp, PCR 468bp and 663bp for the second time for the first time), on sepharose, be easy to differentiate.But not the reference culture of streptococcus mutans group there is no any amplified band (see figure 3).As seen, this PCR reaction pair becomes suis group bacterium and also has specificity, has specificity because of streptococcus mutans and streptococcus sobrinus expanding fragment length again, proves that this PCR reaction can effectively distinguish this two kinds of bacterium.And PCR reaction for the first time is with 10
5The DNA of CFU makes template, visible specific amplification band.And PCR reaction for the second time, only 10
3The DNA of CFU makes template all specific amplification band (seeing Fig. 4-7).N-PCR also can detect streptococcus mutans and the streptococcus sobrinus (seeing Fig. 8 and 9) in the saliva sample.Therefore, the result who carries out PCR at individual research object 1ml saliva sample can be divided into the infectation of bacteria level three levels: 〉=10
5CFU is the hyperinfection object; 〉=10
3-＜10
5CFU is the grade and moderate infection object;＜10
3CFU is for minuent infects object.
Claims (1)
1, the detection method of streptococcus mutans and streptococcus sobrinus in a kind of people's saliva, it comprises the following steps:
A, design primer: the nested polymerase chain reaction that is provided comprises secondary PCR, streptococcus mutans first align anti-primer be 5 '-TAACTACACTTTCGGGTGGCTT and 5 '-GATGTATCAGTATAAGCGCCAG, second align anti-primer be 5 '-AAAGCAGATTCTAATGAATCGA and 5 '-AATGTAAAATTTTGCCATCAGC; Streptococcus sobrinus first align anti-primer be 5 '-GGTATCGTCCAAAATCAATCC and 5 '-TTATCGATACCGTAAGCTGCC, second align anti-primer be 5 '-TGGTATCGTCCAAAATCAATCC and 5 '-AGATTTGCAGTTGGTCAGCATC;
B, bacterial chromosomal dna and salivary chromosome DNA extraction: the extraction that at first is DNA of bacteria: 3ml bacterium culture overnight, 10000rpm is centrifugal, 4 ℃ 10 minutes, remove supernatant, 200 μ lTE damping fluids flushings secondary is resuspended in precipitation in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃ 30 minutes, add 40 μ g Proteinase Ks, and add 10%SDS45 μ l, mixing, 37 ℃ 3 hours, equal-volume phenol, chloroform extracting secondary, dehydrated alcohol deposit D NA centrifugal 10000rpm10 minute, abandons supernatant, 70% washing with alcohol is dissolved in the TE damping fluid; Next is the extraction of salivary chromosome DNA: 1ml saliva, 8000rpm, 4 ℃ centrifugal 15 minutes, remove supernatant, 500 μ lTE damping fluids are washed secondary, precipitation is resuspended in the 200 μ lTE liquid that include the 2mg N,O-Diacetylmuramidase, 37 ℃ 30 minutes, add 4 μ g Proteinase Ks, and add 10%SDS45 μ l, mixing, 37 ℃ 3 hours, equal-volume phenol, chloroform extracting secondary, ethanol sedimentation DNA, centrifugal 1,0000rpm, 10 minutes, abandon supernatant, 70% washing with alcohol is dissolved in the TE damping fluid;
C, PCR for the first time: add two couples of outer primer thp3, thp4, each 50pmol of thp5, thp6 successively, 4 kinds of dNTP mixture 4 μ l, 10 * reaction buffer, 5 μ l, bacterium template DNA 2 μ l, TaqDNA polysaccharase 2.5U, aqua sterilisa 36 μ l, mixing, reaction was by 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, circulate 30 times, last 72 ℃ were extended 5 minutes;
D, PCR for the second time: the PCR product is got 2 μ l for the first time, dilute 10 times after, get 2 μ l and make template, with thp7, thp8, each 50pmol of thp9, thp10, its step is with the PCR first time;
E: electrophoresis: get amplified production 5 μ l, 1.0% agarose gel electrophoresis voltage 80V, the ingot dyeing of 0.5 μ g/ml bromination, with DL2,000 DNA Marker is a standard control, observes amplified band under Ultraviolet Detector.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102440234A (en) * | 2010-09-30 | 2012-05-09 | 上海泛亚生命科技有限公司 | Stationary liquid for preserving human saliva, preparation and application |
| CN103060442A (en) * | 2012-11-21 | 2013-04-24 | 重庆原伦生物科技有限公司 | Double polymerase chain reaction (PCR) rapid detection method of streptococcus mutans |
| CN109825499A (en) * | 2019-04-18 | 2019-05-31 | 华南农业大学 | A kind of extraction reagent, kit and the application and extracting method of cell-bacterial parasite total DNA |
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2005
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102440234A (en) * | 2010-09-30 | 2012-05-09 | 上海泛亚生命科技有限公司 | Stationary liquid for preserving human saliva, preparation and application |
| CN103060442A (en) * | 2012-11-21 | 2013-04-24 | 重庆原伦生物科技有限公司 | Double polymerase chain reaction (PCR) rapid detection method of streptococcus mutans |
| CN109825499A (en) * | 2019-04-18 | 2019-05-31 | 华南农业大学 | A kind of extraction reagent, kit and the application and extracting method of cell-bacterial parasite total DNA |
| CN109825499B (en) * | 2019-04-18 | 2020-11-06 | 华南农业大学 | Extraction reagent and kit for total DNA of cell-parasitic bacteria, application and extraction method |
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