CN101935686A - PCR method of multiple sex propagate pathogene synchronous detection and kit - Google Patents
PCR method of multiple sex propagate pathogene synchronous detection and kit Download PDFInfo
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- CN101935686A CN101935686A CN2009100038008A CN200910003800A CN101935686A CN 101935686 A CN101935686 A CN 101935686A CN 2009100038008 A CN2009100038008 A CN 2009100038008A CN 200910003800 A CN200910003800 A CN 200910003800A CN 101935686 A CN101935686 A CN 101935686A
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Abstract
The invention discloses a single-tube multiple PCR method for synchronous rapid detection of five sex propagate pathogenes (neisseria gonorrhoeae (NG), mycoplasma hominis (MH), mycoplasma genitalium (MG), chlamydia trachomatis (CT) and ureaplasma urealyticum (UU)) and a kit. The method is characterized by amplifying the five pathogenes and performing gel electrophoresis separation and detection by reasonably designing a primer and optimizing the primer concentration combination and PCR condition in the same reaction tube and under the same heat cycle condition. The invention has the characteristics of sensitivity, rapidness, convenience, and the like of the classic PCR, mainly realizes synchronous detection of the five pathogenes, has lower cost, can be used for developing relative kits and is used for clinical diagnosis and epidemiological survey and control.
Description
The PCR method of five kinds of sexually transmitted disease (STD) pathogenic agent of a kind of single tube multiple PCR technique rapid detection (NG, UU, CT, MG and MH), test kit are formed and are used
Technical field
The present invention relates to the PCR method and the test kit of five kinds of sexually transmitted disease (STD) pathogenic agent of a kind of rapid detection, be applicable to the quick clinical diagnosis and the epidemiology survey of sexually transmitted disease (STD).
Background technology
(Sexually Transmitted disease STD) is present common disease frequently-occurring disease to sexually transmitted disease (STD).Traditional sexually transmitted disease (STD) detection method mainly comprises: the separation of pathogenic agent and biological test, electron microscopy, immunofluorescence technique, fluorescent quantitative PCR technique etc., these methods have played vital role in medical diagnosis on disease, but ubiquity significantly not enough, long such as the cycle, complex operation can only detect one or both pathogenic agent at every turn.At present, the multiple Combination infection rate (Shen Jianwei that obviously rises that spreads disease, Sun Xiuqin, Cheng Donge. female genital tract sexually transmitted disease (STD) pathogenic agent polyinfection investigation. Chinese hospital infection magazine [j], 2007, (05) .), using more widely, fluorescent quantitative PCR technique once can only detect a kind of pathogenic agent, if a sample is arranged, detect one or more that whether contain above-mentioned pathogenic agent, then need to carry out repeatedly respectively the PCR reaction, detect the cost height, bring difficulty for the detection and the diagnosis of sexually transmitted disease (STD), the laboratory diagnosis technology obviously lags behind.
At present, the single tube multiple PCR technique that is developing promptly in a reaction tubes, under same reaction conditions two or more different target genes are carried out synchronous amplification, can improve the efficient of PCR so greatly.
We utilize the single tube multiple PCR technique, grope its PCR reaction and resistant to pollution top condition, successfully with detecting U.urealyticum (Ureaplasma urealyticum simultaneously with a processing sample and a pcr amplification, UU), M.homins (mycoplasma hominis, MH), M.genitalium (mycoplasma genitalium, MG), Chlamydia trachomatis (chlamydia trachomatis, CT) and Neisseria gonorrhoeae (Diplococcus gonorrhoeae, NG) five kinds of STD pathogenic agent think that by blind method assay this is the high special detection method of a kind of high-level efficiency.
Summary of the invention
The method that the purpose of this invention is to provide one kind of multiple pathogenic agent synchronous detection that spread through sex intercourse, this method is by the design specialized amplimer, provide the multiplex PCR condition of the synchronous detection of five kinds of pathogenic agent, this is easy to implement the method, can be fast, five kinds of pathogenic agent in the synchronous detection sample easily.
For achieving the above object, the present invention adopts following technical measures: design suitable primer, make between the primer and do not disturb, and have similar amplification condition.The steps include:
A. design the primer that a cover is used for synchronous amplification five kinds of pathogenic agent UU, MH, MG, CT, NG, each primer is at corresponding pathogenic agent high conservative gene, the annealing temperature of different primers is approximate, primer does not have the above complementation of three bases between any two, the PCR product in twos the difference of length greater than 40-50bp, be beneficial to sepharose and separate, the primer sequence of five kinds of pathogenic agent NG, UU, CT, MG and MH sees Table 1:
Table 1: the primer sequence of five kinds of pathogenic agent NG, UU, CT, MG and MH
B. optimize the condition of multiplex PCR: each primer micro-molar concentration (umol/L) optimum combination sees Table 2:
Table 2: each primer micro-molar concentration (umol/L) optimum combination
| NG? | MH? | MG? | CT? | UU? |
| NP1/NP2? | MHP1/MHP2? | MP1/MP2? | CP1/CP2? | UP1/UP2? |
| 0.5? | 0.4? | 0.4? | 0.4? | 1.5? |
C. the target gene synchronous amplification of five kinds of pathogenic agent, the optimum response system is: reaction volume 25ul, Taq enzyme 1.0U, damping fluid: 1xGCbuffer, Mg2+:3.0mmol/L, dNTPs 400umol/L, be reflected on the thermal cycler and carry out, adopt same thermal cycle conditions; After reaction finishes, get 10 μ l PCR products and 0.8 μ l Generuler (Shenzhen is brilliant beautiful) and under 1 * TEB damping fluid, 5-6V/cm condition, run 2% agarose gel electrophoresis 40min, EB dyeing back observations under UVP gel imaging instrument (UVP Inc, the U.S.).
D. the checking of optimal conditions, because it is very little to occur the probability of five kinds of pathogenic agent in same duplicate samples simultaneously, whether the multiplex PCR condition that needs checking to be set up can detect every kind of virus exists individually or simultaneously, therefore, under optimal conditions, utilize reference culture to carry out the heavy pcr amplification of 1-5 respectively, and adopt gel electrophoresis separation detection result.
Fig. 1 is 5 kinds of haplotype standard DNA amplified band electrophoretograms, is single reference culture pathogenic agent optimization experiment result under the multiplex PCR condition.
No. 1 on behalf of No. 2 position 241bp of UU, position 167bp represent No. 3 position 281bp of CT to represent MG
No. 4 on behalf of No. 5 position 390bp of MH, position 334bp represent NG B, M position to represent blank and standard
Fig. 2 is 5 kinds of mutual mixed type standard DNA amplified band electrophoretograms, is various hybrid standard bacterial strain pathogenic agent optimization experiment results under the multiplex PCR condition.
On behalf of UU 241bp, 167bp represent CT 281bp to represent MG
On behalf of MH 390bp, 334bp represent NG
No. 1 on behalf of No. 2 positions of UU/CT, the position represent No. 3 positions of UU/CT/MH to represent UU/NG
4, on behalf of No. 5 positions of UU/CT/MH/NG, No. 7 positions represent No. 6 positions of UU/MH to represent UU/CT/NG
No. 8 on behalf of UU/CT/MG/MH/NG B, M position, the position represent blank and standard
Below in conjunction with accompanying drawing the present invention is described in further detail:
Reference culture source NG, MH, MG, all (NG:ATCC 49226 available from Guangdong Province's Center for Disease Control for the UU reference culture; MH:ATCC 14027; MG:ATCC 33530; UU:ATCC 27813), the CT reference culture is available from Guangdong Da An genome company (mixture of ATCC 25922 and ATCC 25923).
2. the hybrid standard bacterial strain is made various reference cultures with 1: 1 ratio preparation standard compound sample.
3.DNA template preparation adopt the vibration mixing, centrifugal, add alkaline bleach liquor cleavage liquid, 95 ℃ of heating, be cooled to add neutralizer after 4 ℃, the alkaline lysis of centrifuging and taking supernatant liquor prepares pcr template again.
4.PCR design of primers (table 3) is totally 5 pairs of primers, the target gene of each primer is corresponding pathogenic agent high conservative regional gene, the annealing temperature of primer is 56 ℃-64 ℃, primer does not have the above complementation of three bases between any two, the PCR product in twos the difference of length greater than 40-50bp, be beneficial to sepharose and separate, primer is by oneself design and synthetic;
5.PCR condition in 25 μ l PCR reaction volumes, contains 1 * GC buffer I, 400 μ mol/L dNTP, 1.0U TaKaRa LA Taq, primer and target DNA concentration see Table 4.PCR carries out on the amplification instrument.Thermal circulation parameters (seeing Table 5) is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min then, 60 ℃ of 1min, 35 circulations of 72 ℃ of 2min, last 72 ℃ are extended 5min.
3. after the amplification of amplified production electrophoresis, get 10 μ l PCR products and 0.8 μ l Generuler (Shenzhen is brilliant beautiful) and under 1 * TEB damping fluid, 5-6V/cm condition, run 2% agarose gel electrophoresis 40min, EB dyeing back is in UVP gel imaging instrument (UVP Inc, the U.S.) observations under, the substance pcr amplification product is seen accompanying drawing 1, and the single tube multiplex PCR is seen accompanying drawing 2.
The primer sequence of each pathogenic agent of table 3, amplified target gene and amplified production size (previous in the every pair of primer is upstream primer, and back one is downstream primer)
Table 4 multi-PRC reaction detects the condition of STD pathogenic agent
Table 5 PCR thermal cycle conditions
The present invention compared with prior art has following advantage and effect:
1. the result is accurate
2. detection speed is fast, and step is simple, can detect a plurality of pathogenic micro-organisms simultaneously
3. expense is low
Embodiment
Below in conjunction with concrete case study on implementation, further set forth the present invention.Should be appreciated that these case study on implementation only to be used to the present invention is described and be not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following case, the condition of advising usually according to manufacturer.
The clinical suspicious sample of the preparation of 1 sample source and pcr template derives from Guangdong Provincial TCM Hospital Zhuhai hospital outpatient in April, 2003～2003 year May, and totally 211 parts, 116 parts is women's cervical secretions, and 95 parts is the male urethra swab.Adopt the vibration mixing, centrifugal, add alkaline bleach liquor cleavage liquid, 95 ℃ of heating, be cooled to add neutralizer after 4 ℃, the alkaline lysis of centrifuging and taking supernatant liquor prepares pcr template again.
2 design of primers adopt Primer Primer5 and Oligo5.0 software design primer sequence, and by qualitatively screening until obtaining the suitableeest primer.The size (bp) of the primer, 5 ' of each pathogenic agent → 3 ' sequence, amplified target gene and amplimer is asked for an interview table 6.These primers are designed voluntarily by the author.
3 PCR conditions and product detect in 25 μ l PCR reaction volumes, contain 1 * GC buffer I, 400 μ mol/L dNTP, 1.0U TaKaRaLA Taq, and primer and target DNA concentration see Table 7.PCR carries out on PE-2400 increases instrument (Gene Amp2400, Perkin Elmer).Thermal circulation parameters is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min then, 60 ℃ of 1min, 35 circulations of 72 ℃ of 2min, last 72 ℃ are extended 5min.After the amplification, get 10 μ l PCR products and 0.8 μ l Generuler (Shenzhen crystalline substance is beautiful) and under 1 * TEB damping fluid, 5-6V/cm condition, run 2% agarose gel electrophoresis 40min, EB dyeing back observations under UVP gel imaging instrument (UVP Inc, the U.S.).
4 results detect as gold standard and multiplex PCR for the outpatient service sample of STD adds another kind of detection technique with quantitative fluorescent PCR by 211 examples are doubted, the positive findings that 2 kinds of methods the obtain rate of coincideing reaches 100%, and what concrete individual event and polyinfection coincide rate relatively sees Table 8
Each pathogenic agent of table 6 and interior target primer sequence amplified target gene and amplified production size
Table 7 multi-PRC reaction detects the condition of STD pathogenic agent
The comparison of the identical rate of table 8 136 routine gold standard method male pathogenic agent and multiplex PCR result
Claims (1)
1. the PCR method and the test kit of one kind of multiple sexually transmitted disease (STD) pathogenic agent synchronous detection, this method comprises following reagent and step:
A. be designed for the primer of synchronous amplification five kinds of pathogenic agent NG, UU, CT, MG and MH, each primer is the high conservative gene at corresponding pathogenic agent, the annealing temperature of primer is 56 ℃～64 ℃, primer does not have the above complementation of three bases between any two, pcr amplification product in twos the difference of length greater than 40～50bp;
B. the primer sequence of five kinds of pathogenic agent NG, UU, CT, MG and MH is:
C. the PCR of five kinds of pathogenic agent NG, UU, CT, MG and MH gene synchronous detection, reaction volume 25ul, Taq enzyme 1.0U, damping fluid: lx GC, Mg2+:3.0mmol/L, dNTPs:400umol/L, primer micro-molar concentration (umol/L) is combined as:
Be reflected on the thermal cycler and carry out, thermal circulation parameters is: 94 ℃ of pre-sex change 3min, and 94 ℃ of 1min, 60 ℃ of 1min, 35 circulations of 72 ℃ of 2min then, last 72 ℃ are extended 5min
D. after the amplification, get 10 μ l PCR products and 0.8 μ l Generuler (Shenzhen crystalline substance is beautiful) and under 1 * TEB damping fluid, 5-6V/cm condition, run 2% agarose gel electrophoresis 40min, EB dyeing back observations under UVP gel imaging instrument (UVP Inc, the U.S.).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888464A (en) * | 2012-10-25 | 2013-01-23 | 苏州华益美生物科技有限公司 | Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof |
| CN108517350A (en) * | 2018-04-09 | 2018-09-11 | 中国人民解放军陆军军医大学第附属医院 | Library kit is built in multiple pathogens targeting |
| CN109825615A (en) * | 2019-01-09 | 2019-05-31 | 中生方政生物技术股份有限公司 | Primer, probe, kit and the detection method of chlamydia trachomatis, gonococcus and urea substance Multiple detection |
-
2009
- 2009-01-23 CN CN2009100038008A patent/CN101935686A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888464A (en) * | 2012-10-25 | 2013-01-23 | 苏州华益美生物科技有限公司 | Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof |
| CN102888464B (en) * | 2012-10-25 | 2014-04-02 | 苏州华益美生物科技有限公司 | Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof |
| CN108517350A (en) * | 2018-04-09 | 2018-09-11 | 中国人民解放军陆军军医大学第附属医院 | Library kit is built in multiple pathogens targeting |
| CN108517350B (en) * | 2018-04-09 | 2019-07-16 | 中国人民解放军陆军军医大学第一附属医院 | Library kit is built in multiple pathogens targeting |
| CN109825615A (en) * | 2019-01-09 | 2019-05-31 | 中生方政生物技术股份有限公司 | Primer, probe, kit and the detection method of chlamydia trachomatis, gonococcus and urea substance Multiple detection |
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Application publication date: 20110105 |