CN106520986A - Quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously - Google Patents
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Abstract
The invention discloses a quintuple PCR (polymerase chain reaction) detection method capable of detecting multiple pathogens simultaneously. According to the method, firstly, genes with conservative sequences of staphylococcus aureus, pseudomonas aeruginosa, pasteurella multocida, bordetella bronchiseptica and mycoplasma pulmonis are screened out and taken as targets for PCR detection, specific primers of corresponding conservative sequences are synthesized and amplified respectively, five pairs of primers are placed in one PCR system, and the quintuple PCR detection method capable of detecting five pathogens once from a sample directly is established through optimization of each item. Compared with a conventional PCR detection method, the detection method has the characteristics of rapidness, sensitivity, simplicity, convenience and accuracy, and the requirements for detection people and the detection cost are reduced.
Description
Technical field
The present invention relates to a kind of PCR detection method of biological technical field, specifically one kind can detect golden yellow Portugal simultaneously
Five heavy PCR detection methods of grape coccus, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises.
Background technology
Existing national standard《Laboratory animal microbiology grade and monitoring》To pathogen in GB 14922.2-2011
Detection method is based on traditional separation and Culture, biochemical identification, serum type analysis and special identification experiment.In clinical practice
In, staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises are experiments
The common important pathogen body of animal, and easily mixed infection.For example separation and Culture, immunological testing etc. are time-consuming takes for traditional diagnostic techniquess
Power, is unsuitable for clinical quick diagnosis, and being also unsuitable for the present situation traditional method of large-scale Epidemiological study, to there is process loaded down with trivial details,
Time-consuming, recall rate is low and the defect such as missing inspection.In order to meet the quick detection of the multiple pathogens of large sample, be badly in need of it is a kind of it is quick,
The high multi-PCR detection method of easy, accuracy rate.
The content of the invention
It is an object of the invention to provide a kind of while detecting five heavy PCR detection methods of multiple pathogens, its step is simple
Single, detection is quick, and accuracy is high, it is easy to standardization, overcomes the deficiencies in the prior art.
Idea of the invention is that such.With PCR nucleic acid Calibration Technology successfully the fields such as clinical medicine should
With, set up multiplex PCR new technique detection be possibly realized.Multiplex PCR (multiplex Polymerase Chain Reaction)
It is to be improved on the basis of regular-PCR, in a PCR reaction system, adds multipair specific primer, for multiple DNA mould
The zones of different of plate or same template amplifies the round pcr of multiple purpose fragments.Which can be in same PCR reaction systems simultaneously
The specific primer of multiple pathogens is added, is entered performing PCR amplification, be can be used for while detecting multiple pathogens or different shaped cause of disease
Body.Based on this, the present invention can just detect the target of five kinds of pathogen in sample in line with disposable detection, first from having delivered
Filter out in document with staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and lung
The gene of the conservative sequence of mycoplasma, used as the target spot of PCR detections, the specificity for being respectively synthesized amplification correspondence conserved sequence draws
Thing, 5 pairs of primers are placed in a PCR reaction system simultaneously, and by every optimization, foundation is directly disposably detected from sample
Five heavy PCR detection methods of five kinds of pathogen.The present invention is also drawn materials by detecting that feces are improved, and is further realized using five weight
The purpose of PCR method quick detection actual sample.
Specifically, the present invention's is a kind of while five heavy PCR detection methods of detection multiple pathogens, comprise the following steps：
(1) filter out with staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella Podbielniak bar
The gene of the conservative sequence of bacterium and mycoplasma pulmonises, as the target spot of PCR detections, is respectively synthesized the spy of amplification correspondence conserved sequence
Specific primer；
(2) 5 pairs of primers are placed in a PCR reaction system simultaneously, amplify genes of interest fragment；
(3) by agarose gel electrophoresiies Testing and appraisal pcr amplification product；Wherein, expand staphylococcus aureuses upstream
, as shown in table 1a, the sequence of downstream primer is as shown in table 1b for the sequence of primer；The sequence such as table of amplification bacillus pyocyaneus forward primer
Shown in 1c, the sequence of downstream primer is as shown in table 1d；Amplification mycoplasma pulmonises forward primer sequence as shown in table 1e, downstream primer
Sequence as shown in table 1f；Amplification pasteurella multocida forward primer sequence as shown in table 1g, the sequence such as table of downstream primer
Shown in 1h；Amplification Bordetella bordetella bacilli forward primer sequence as shown in table 1i, the sequence such as table 1j of downstream primer
It is shown.1 is shown in Table specifically.
Table 1：Primer sequence and PCR primer
In the present invention, step (1) is as the annealing temperature to primer, fragment length etc. are required, final to select golden yellow Portugal
The nuc genes of grape coccus, the LasI genes of bacillus pyocyaneus, the KMT1 genes of pasteurella multocida, Bordetella Podbielniak bar
The 16S rRNA gene design specific primers of the Fla genes and mycoplasma pulmonises of bacterium.
In the present invention, step (2), PCR reaction systems are 50 μ l, including：25 μ l of Mix, 7 μ l of primer, Staphylococcus aureus
Bacterium, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer respectively for 1 μ l, 1 μ l,
0.5 μ l, 0.5 μ l, 0.5 μ l, the every kind of 1 μ l DNA of DNA, remaining sterilized water are supplied；Reaction condition：94 DEG C of denaturations 5min；Into
94 DEG C of 30s, 60 DEG C of 30s, the circulation of 72 DEG C of 30s, totally 30 circulations；72 DEG C extend 7min eventually.
In the present invention, step (3), PCR primer in 2.5% agarose gel, 120V electrophoresis 30min.
In the present invention, first select manually to mix pathogen positive sample checking system power of test before actually detected sample, then
Sample feces to be detected and tracheal secretion are taken, the DNA of tested pathogen is extracted, as template, five weight PCR detections is carried out, entirely
Process only needs 4h；And drawn materials by detecting that feces are improved, further up to using the quick Non-invasive detection of five weights PCR method
The purpose of noinvasive actual sample.And the detection method of routine needs culture medium inoculated, typically need 24h to grow bacterium colony, then biochemistry connects
Kind, this process time is longer, and high to the skill set requirements of testing staff.The more conventional method of the present invention shortens detection time,
Can be further up to quick detection and the purpose of standardized testing.
The present invention obtains following beneficial effect：
(1) more conventional PCR detection method, five weight PCR detection methods of the invention have quick, sensitive, easy, accurate
The characteristics of, while reducing the requirement to testing staff, detection platform and testing cost, improve detection efficiency.
(2) party's law system is drawn materials by detecting that feces are improved, further up to using the quick noinvasive of five weights PCR method
The purpose of detection noinvasive actual sample.
Description of the drawings
Fig. 1 is staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and pulmonary branches
The optimum results of five heavy PCR system primer concentrations of substance；Wherein：M is 100bp DNA Ladder；Swimming lane 1-4：Kill Pasteur more
Bacillus, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer are respectively 1 μ l, 0.5 μ l, 0.25 μ l, 0.125 μ l.
Fig. 2 is staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and pulmonary branches
The optimum results of five heavy PCR system annealing temperatures of substance；Wherein：M is 100bp DNA Ladder；Swimming lane 1：56℃；Swimming lane
2：58℃；Swimming lane 3：60℃；Swimming lane 4：62℃.
Fig. 3 is staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and pulmonary branches
The testing result of five heavy PCR artificial samples of substance；Wherein：M is 100bp DNA Ladder；Swimming lane 1：The five heavy PCR positives are right
According to；Swimming lane 2：Five heavy PCR negative controls；Swimming lane 3：Feces negative control；Swimming lane 4：Staphylococcus aureuses；Swimming lane 5：Green pus bar
Bacterium；Swimming lane 6：Staphylococcus aureuses and bacillus pyocyaneus；Swimming lane 7：Tracheal secretion negative control；Swimming lane 8：Kill Pasteur's bar more
Bacterium；Swimming lane 9：Bordetella bordetella bacilli；Swimming lane 10：Mycoplasma pulmonises；Swimming lane 11：Pasteurella multocida, bronchus deteriorated blood
Property bordetella bacilli and mycoplasma pulmonises；Swimming lane 12：Staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella
Bordetella bacilli and mycoplasma pulmonises.
Fig. 4 is staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and pulmonary branches
The testing result of five heavy PCR samples of substance；Wherein：M is 100bp DNA Ladder；Swimming lane 1：Five heavy PCR positive controls；Swimming
Road 2：Five heavy PCR negative controls；Swimming lane 3-17：The feces and tracheal secretion of sample.
Specific embodiment
The present embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and mistake
Journey, but protection scope of the present invention is not limited to following embodiments.
1 staphylococcus aureuses of embodiment, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and lung
The optimization of five heavy PCR system annealing temperatures of mycoplasma
(1) sample pre-treatments
By the staphylococcus aureuses of standard, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and lung
Mycoplasma is cultivated in corresponding culture medium or culture fluid respectively, wherein staphylococcus aureuses, bacillus pyocyaneus culture 24h, many
Pasteurellosis bacilluss, Bordetella bordetella bacilli culture 36h, mycoplasma pulmonises culture 7d are killed, bacterium solution is collected or bacterium colony is extracted corresponding
DNA sample.
(2) DNA of bacteria is extracted
The present invention extracts DNA using bacterial genomes DNA extraction kit, and step is as follows：
1. above-mentioned 5 kinds of pathogen bacterium solutions or bacterium colony are dissolved in into buffer GA, concussion suspends；
2. 20 μ l Proteinase K solution are added, are mixed, add 220 μ l buffer GB, shake 15s, 70 DEG C of 10min,
220 μ l dehydrated alcohol, fully shaking are added to mix；
3. solution and precipitation are all added in an adsorption column, 12000rpm centrifugation 30s abandon waste liquid；
4. add 500 μ l buffer GD, 12000rpm centrifugation 30s to adsorption column, abandon waste liquid；
5. add 600 μ l buffer PW, 12000rpm centrifugation 30s to adsorption column, abandon waste liquid；It is repeated once；
6. precipitation, 30 μ l TE dissolvings are collected.
(3) multi-PRC reaction Establishing
In PCR reaction systems, especially in multi-PRC reaction system, primer concentration and annealing temperature are crucial shadows
The factor of sound.The present invention is tested as follows in order to find the primer concentration and annealing temperature of suitable five weights PCR system：
1. the optimization of primer concentration：Reaction system：Staphylococcus aureuses, bacillus pyocyaneus upstream and downstream primer are respectively 1 μ l, many
Kill pasteurellosis bacilluss, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer respectively for 1 μ l, 0.5 μ l, 0.25 μ l, 0.125
The every kind of 1 μ l DNA of 25 μ l of μ l, Mix, DNA, remaining complements to 50 μ l with sterilized water；Reaction condition：94 DEG C of denaturations 5min；Enter
Enter 94 DEG C of 30s, 60 DEG C of 30s, the circulation of 72 DEG C of 30s, totally 30 circulations；72 DEG C extend 7min eventually；10 μ l of PCR primer are 2.5%
In agarose gel, 120V electrophoresis 30min.As a result as Fig. 1 shows that 2 swimming lanes are preferable.
2. the optimization of annealing temperature：PCR reaction systems are 50 μ l, including：25 μ l of Mix, 7 μ l of primer, Staphylococcus aureus
Bacterium, bacillus pyocyaneus upstream and downstream primer are respectively 1 μ l, and pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises are upper and lower
Trip primer is respectively 0.5 μ l, and the every kind of 1 μ l DNA of DNA, remaining sterilized water are supplied.Annealing temperature is only respectively set to by reaction condition
56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C, remaining is constant.As a result as Fig. 2 shows that 3 swimming lanes are preferable.
(4) result
PCR reaction systems are 50 μ l, including：25 μ l of Mix, 7 μ l of primer, staphylococcus aureuses, bacillus pyocyaneus, kill more
Pasteurellosis bacilluss, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer are respectively 1 μ l, 1 μ l, 0.5 μ l, 0.5 μ l, 0.5 μ
The every kind of 1 μ l DNA of l, DNA, remaining sterilized water are supplied.Reaction condition：94 DEG C of denaturations 5min；Into 94 DEG C of 30s, 60 DEG C of 30s,
The circulation of 72 DEG C of 30s, totally 30 circulations；72 DEG C extend 7min eventually.
2 staphylococcus aureuses of embodiment, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and lung
The detection of five heavy PCR artificial samples of mycoplasma
(1) sample pre-treatments
Take the sample feces and tracheal secretion of animal to be detected.
(2) DNA of bacteria is extracted
With embodiment 1
(3) multi-PRC reaction Establishing
PCR reaction systems are 50 μ l, 25 μ l of Mix, 7 μ l of primer, staphylococcus aureuses, bacillus pyocyaneus, kill Pasteur's bar more
Bacterium, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer are respectively 1 μ l, 1 μ l, 0.5 μ l, 0.5 μ l, 0.5 μ l, DNA
Respectively 5 μ l of positive control, 0 μ l of negative control, sample feces and each 2 μ l of tracheal secretion, remaining sterilized water complement to 50 μ l.
Reaction condition：94 DEG C of denaturations 5min；Into 94 DEG C of 30s, 60 DEG C of 30s, the circulation of 72 DEG C of 30s, totally 30 circulations；72 DEG C of ends prolong
Stretch 7min.10 μ l of PCR primer in 2.5% agarose gel, 120V electrophoresis 30min.
(4) result
Staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises
The testing result of five heavy PCR samples is as shown in Figure 3：1 positive control；2 negative controls；3rd, the feminine gender of 7 feces and tracheal secretion
Control；4 staphylococcus aureuses；5 bacillus pyocyaneus；6 staphylococcus aureuses and bacillus pyocyaneus；8 pasteurella multocidas；9 gas
Pipe septicemic bordetella bacilli；10 mycoplasma pulmonises；11 pasteurella multocidas, Bordetella bordetella bacilli and mycoplasma pulmonises；12
Staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises.Seen by result
Go out, the five heavy PCR detection system can detect staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella
Bordetella bacilli and mycoplasma pulmonises.
3 staphylococcus aureuses of embodiment, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and lung
The detection of five heavy PCR samples of mycoplasma
(1) sample pre-treatments
Take the sample feces and tracheal secretion of animal to be detected.
(2) DNA of bacteria is extracted
With embodiment 1
(3) multi-PRC reaction Establishing
PCR reaction systems are 50 μ l, 25 μ l of Mix, 7 μ l of primer, staphylococcus aureuses, bacillus pyocyaneus, kill Pasteur's bar more
Bacterium, Bordetella bordetella bacilli and mycoplasma pulmonises upstream and downstream primer are respectively 1 μ l, 1 μ l, 0.5 μ l, 0.5 μ l, 0.5 μ l, DNA
Respectively 5 μ l of positive control, 0 μ l of negative control, sample feces and each 2 μ l of tracheal secretion, remaining sterilized water complement to 50 μ l.
Reaction condition：94 DEG C of denaturations 5min；Into 94 DEG C of 30s, 60 DEG C of 30s, the circulation of 72 DEG C of 30s, totally 30 circulations；72 DEG C of ends prolong
Stretch 7min.10 μ l of PCR primer in 2.5% agarose gel, 120V electrophoresis 30min.
(4) result
Staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises
The testing result of five heavy PCR samples is as shown in Figure 4：1 positive control；2 negative controls；The feces of 3-17 samples and trachea secretion
Thing.Positive control and negative control be good, and sample is detected as feminine gender, consistent with the testing result of traditional method.
SEQUENCE LISTING
<110>Hebei Medical University
<120>It is a kind of at the same detect multiple pathogens five heavy PCR detection methods
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> Staphylococcus aureus
<400> 1
cacctgaaac aaagcatcct aa 22
<210> 2
<211> 21
<212> DNA
<213> Staphylococcus aureus
<400> 2
tatacgctaa gccacgtcca t 21
<210> 3
<211> 21
<212> DNA
<213> Pseudomonas aeruginosa
<400> 3
atgatcgtac aaattggtcg g 21
<210> 4
<211> 18
<212> DNA
<213> Pseudomonas aeruginosa
<400> 4
gtcatgaaac cgccagtc 18
<210> 5
<211> 23
<212> DNA
<213> Bordetella bronchiseptica
<400> 5
aggctcccaa gagagaaagg ctt 23
<210> 6
<211> 16
<212> DNA
<213> Bordetella bronchiseptica
<400> 6
tggcgcctgc cctatc 16
<210> 7
<211> 20
<212> DNA
<213> Mycoplasma pneumoniae
<400> 7
agcgtttgct tcactttgaa 20
<210> 8
<211> 20
<212> DNA
<213> Mycoplasma pneumoniae
<400> 8
gggcatttcc ctccctagct 20
<210> 9
<211> 20
<212> DNA
<213> Pasteurella multocida
<400> 9
gtgagtgggc ttctcggtag 20
<210> 10
<211> 20
<212> DNA
<213> Pasteurella multocida
<400> 10
gctgtaaacg aactcgccac 20
Claims (4)
1. a kind of at the same detect multiple pathogens five heavy PCR detection methods, it is characterised in that comprise the following steps：
（1）Filter out with staphylococcus aureuses, bacillus pyocyaneus, pasteurella multocida, Bordetella bordetella bacilli and
The gene of the conservative sequence of mycoplasma pulmonises, as the target spot of PCR detections, is respectively synthesized the specificity of amplification correspondence conserved sequence
Primer；
（2）5 pairs of primers are placed in a PCR reaction system simultaneously, genes of interest fragment is amplified；
（3）By agarose gel electrophoresiies Testing and appraisal pcr amplification product；Wherein, expand golden yellow Portugal
, as shown in 1 a of table, the sequence of downstream primer is as shown in 1 b of table for the sequence of grape coccus forward primer；On amplification bacillus pyocyaneus
, as shown in 1 c of table, the sequence of downstream primer is as shown in 1 d of table for the sequence of trip primer；The sequence of amplification mycoplasma pulmonises forward primer
As shown in 1 e of table, the sequence of downstream primer is as shown in 1 f of table；The sequence such as 1 g institutes of table of amplification pasteurella multocida forward primer
Show, the sequence of downstream primer is as shown in 1 h of table；The sequence such as 1 i institutes of table of amplification Bordetella bordetella bacilli forward primer
Show, the sequence of downstream primer is as shown in 1 j of table；.
2. according to claim 1 five heavy PCR detection method, it is characterised in that step（1）In,
The gene of the conservative sequence of staphylococcus aureuses is nuc genes, the gene of the conservative sequence of bacillus pyocyaneus is
LasI genes, the gene of the conservative sequence of pasteurella multocida be KMT1 genes, Bordetella bordetella bacilli it is conservative
Property sequence gene be Fla genes, the conservative sequence of mycoplasma pulmonises gene be 16S rRNA genes.
3. according to claim 1 five heavy PCR detection method, it is characterised in that step（2）In, PCR reaction systems are
50ml, including：Mix 25ml, primer 7ml, primer initial concentration be 10mM, every primer staphylococcus aureus, green pus bar
Bacterium, pasteurella multocida, Bordetella bordetella bacilli and mycoplasma pulmonises add volume be respectively 1ml, 1ml, 0.5ml,
0.5ml, 0.5ml, remaining sterilized water are supplied；Reaction condition is：94℃ 5 min；94 DEG C of 30 s, 60 DEG C of 30s, 72 DEG C of 30s,
Totally 30 circulations； 72℃ 7 min.
4. according to claim 1 five heavy PCR detection method, it is characterised in that step（3）In, PCR primer is in 2.5% fine jade
In sepharose, 120 V electrophoresis, 30 min.
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| CN108004338A (en) * | 2017-12-22 | 2018-05-08 | 杭州医学院 | Detect the Primer composition and application and the product and detection SPF mouse pathogen methods using it of SPF mouse pathogens |
| CN108004338B (en) * | 2017-12-22 | 2021-03-12 | 杭州医学院 | Primer composition for detecting SPF mouse pathogenic bacteria, application and product using primer composition and method for detecting SPF mouse pathogenic bacteria |
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