CN109750115A - The quickly dual qPCR method of detection chlamydia trachomatis and primer and probe - Google Patents
The quickly dual qPCR method of detection chlamydia trachomatis and primer and probe Download PDFInfo
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- CN109750115A CN109750115A CN201910153964.2A CN201910153964A CN109750115A CN 109750115 A CN109750115 A CN 109750115A CN 201910153964 A CN201910153964 A CN 201910153964A CN 109750115 A CN109750115 A CN 109750115A
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Abstract
The present invention provides a kind of dual qPCR method of quickly detection chlamydia trachomatis and primer and probes, include the following steps: (1) early-stage preparations program；(2) nucleic acid extraction and purifying；(3) primer/probe design, synthesis, label；(4) qPCR amplification and result judgement；(5) Monitoring lower-cut and amplification range of linearity test；(6) diagnostic sensitivity, specificity calculate；(7) statistical method；The beneficial effects of the present invention are: Monitoring lower-cuts down to 2copies/PCR, the amplification range of linearity reaches 1.0 × 102-1.0 × 108copies/mL, diagnostic sensitivity and specificity to chlamydia trachomatis are respectively 100.0% (134/134), 99.3% (1142/1150), from sample treatment to report result≤2.0 hour；This method patent simplicity, quick, high sensitivity, specificity are good, and the diagnosis capability of chlamydia trachomatis infection not only can be improved, can also be achieved quick detection, gain time precisely to treat as early as possible.
Description
[technical field]
The present invention relates to a kind of dual qPCR method of chlamydia trachomatis, more particularly to a kind of quickly detection chlamydia trachomatis are double
Weight qPCR method and primer and probe.
[background technique]
Chlamydia trachomatis (Chlamydia trachomatis, CT) is a kind of obligate parasitic cause of disease intracellular, different serum
Type can cause different disease and complication, including reproductive tract infection (serotype D-K), trachoma (serotypes A-C) and venereal disease
Poradenia (serotype L1-L3) [1-3].As the most common cause of disease that spreads through sex intercourse, chlamydia trachomatis can cause serious
Pelvic inflammatory disease, ectopic pregnancy and tubal infertility [4] bring huge pressure and medical treatment to consume to family and society.It is annual complete
Increase chlamydia trachomatis urogenital infections case about 92,000,000, about 50% male the infected and 70% within the scope of ball newly
Female-cases do not show clinical symptoms, cause a large amount of chlamydia trachomatis infection person obtain not in time diagnosis and regular treatment
[5], continue to spread the disease to become the infection sources.
Cell culture because its it is good specificity due to be considered as chlamydia trachomatis detection goldstandard [6].But due to it
It is at high cost, time-consuming, technical difficulty is big, sensitivity is low, be not re-used as the conventional detection means [7] of chlamydia trachomatis.At present
The serological technique of many substitutions, as enzyme-linked immunosorbent assay (ELISA) and immune colloidal gold technique (ICGT) can be significantly
Shorten detection time, but is not suitable for diagnosing acute CT infection [8].Nucleic acid amplification tests (NAATs) with its high sensitivity, Gao Te
Anisotropic and quick feature becomes the chlamydia trachomatis infection diagnostic method [9,10] that Clinical microorganism laboratory is most recommended.It is true
On, real-time fluorescence quantitative PCR (quantitative Real-time PCR, qPCR) and multiple qPCR (multiplex qPCR)
Report time can be foreshortened in 2 hours [11,12] by the appearance Deng modern round pcr.However, really can be used for trachoma clothing
There is highly sensitive and high specific qPCR method still to lack for substance detection, and which results in the leakages of chlamydia trachomatis infection
It examines and mistaken diagnosis.
[summary of the invention]
It is a kind of obligate intracellular present invention aims at chlamydia trachomatis (Chlamydia trachomatis, CT) is solved
Parasitic cause of disease, different serotype can cause different disease and complication, including reproductive tract infection (serotype D-K), husky
Eye (serotypes A-C) and lymphogranuloma venereum (serotype L1-L3) [1-3].As the most common cause of disease that spreads through sex intercourse, trachoma clothing
Substance can cause serious pelvic inflammatory disease, ectopic pregnancy and tubal infertility [4], bring huge pressure to family and society
It is consumed with medical treatment, increasingly developed instantly in accurate medicine, existing invention or reagent have been unable to satisfy demand and one kind for providing
It is able to achieve the dual qPCR method of quick detection chlamydia trachomatis and primer and probe for precisely diagnosing and being suitble to population of China.
The present invention is achieved through the following technical solutions: a kind of quickly detection dual qPCR method of chlamydia trachomatis,
Include the following steps:
(1) early-stage preparations program:
Research sample: leaving and taking the Urogenital Specimens several pieces of sexually transmitted disease patient at random, including male urethra is wiped
Swab and vaginal swab several pieces in sub- several pieces, women uterine neck are evaluated for method.
Instrument and reagent:
1. instrument for extracting nucleic acid (ExPure20) and its mating paramagnetic particle method extraction purification reagent；
2. fluorescence quantitative PCR instrument (LightCycler480)；Multiple fluorescence quantitative PCR system
(PlatinumTMMultiplex PCR Master Mix)；
3. PCR product is sequenced；
(2) nucleic acid extraction and purifying:
1. original sample nucleic acid extraction: carrying out nucleic acid extraction with genital tract sample nucleic acid extracts kit, extraction operation is tight
Lattice are carried out according to instrument and matched reagent SOP；
2. nucleic acid-templated sucking after extraction purification is managed without DNA enzymatic EP, be stored in -86 DEG C freeze it is spare.
(3) primer/probe design, synthesis, label, from ncbi database, download the chlamydia trachomatis blood announced respectively
Clear type D-K bacterial strain cryptic plasmid and 23S rRNA gene order, carry out sequence alignment with DNAMAN, avoid region of variability or catastrophe point
Afterwards, primer and probe synthesis and the label table of each gene are separately designed are as follows:
(4) qPCR amplification and result judgement:
1. multiple qPCR system: 2 × PlatinumTM25.0 μ L of Multiplex PCR Mix, 50 μM of positive anti-primer are each
Each 0.5 μ L of 0.3 μ L, 10 μM of probe, 20.0 μ L of template, moisturizing to 50.0 μ L of final volume；
2. Amplification: 95 DEG C of 2.5min+ (95 DEG C of 10s+60 DEG C of 30s) × 45 select the channel 510nm and 580nm in 60
DEG C detection fluorescence；
3. qualification result judges: the amplification curve of appraisal mark object is in serpentine, and CT value < 37 is judged to the positive；Amplification curve
In serpentine, 37≤CT value < 40 then carries out repeating to detect twice again, if amplification curve remains as serpentine, CT value still < 40,
Also it is judged to the positive；Otherwise it is judged to feminine gender；
4., should when any one or two in two appraisal mark objects are positive findings in multiple qPCR reaction
Sample is judged to positive sample；
(5) Monitoring lower-cut and amplification range of linearity test:
1. the high concentration chlamydia trachomatis nucleic acid template after concentration is carried out 10 times of gradient dilutions with ultrapure water, concentration is made
For 1.0 × 102-1.0 × 108copies/mL gradient concentration template, it to be used for PCR amplification；The minimum addition template of sun can be sentenced
Number is used as Monitoring lower-cut；Using the good minimum and maximum concentration of linear relationship as expanding the range of linearity；
(6) diagnostic sensitivity, specificity calculate:
1. original sample qualification result judges: multiple qPCR method and substance commercial kit are positive, then are judged to kidney-Yang
Property；Multiple qPCR method is positive, and substance commercial kit is negative, is sequenced through an amplified production generation, with mark strain homology >=90%；
Also true positives are judged to；Otherwise, it is judged to false positive；
2. multiple qPCR method is negative, substance commercial kit is negative, then is judged to true negative；Multiple qPCR method is negative, single
Weight commercial kit is positive, is sequenced through amplified production, with mark strain homology >=90%, is judged to false negative；Otherwise, it is also judged to true
It is negative；
3. diagnostic sensitivity=true positives number ÷ (true positives number+false negative number) × 100%；Specificity=true negative
Number ÷ (true negative number+false positive number) × 100%；
(7) statistical method: carrying out student's t check analysis with SPSS to all data, and P < 0.05 is to have statistical difference, and P <
0.01 is to have significant statistical difference.
Further, the sequence of chlamydia trachomatis cryptic plasmid and 23S rRNA genetic test primer and probe
With label table are as follows:
Further, the nt:1755-1880 segment of Simultaneous detection of Chlamydia trachomatis cryptic plasmid and 23S rRNA gene
Nt:51-169 segment, the primer and probe are determined after China's Distribution strain screening, and region of variability or catastrophe point are avoided
It designs.
Further, the dual qPCR reagent of chlamydia trachomatis is quickly detected:
(1) multiple qPCR system: 2 × PlatinumTM25.0 μ L of Multiplex PCR Mix, 50 μM of positive anti-primer
Each 0.5 μ L of each 0.3 μ L, 10 μM of probe, 20.0 μ L of template, moisturizing to 50.0 μ L of final volume；
(2) Amplification: 95 DEG C of 2.5min+ (95 DEG C of 10s+60 DEG C of 30s) × 45 select the channel 510nm and 580nm in 60
DEG C detection fluorescence.
Further, the dual qPCR method of chlamydia trachomatis, qualification result judgment method are quickly detected are as follows:
(1) amplification curve of appraisal mark object is in serpentine, and CT value < 37 is judged to the positive；Amplification curve be in serpentine, 37≤
CT value < 40 then carries out repeating to detect twice again, if amplification curve remains as serpentine, CT value still < 40 is also judged to the positive；It is no
Then it is judged to feminine gender；
In multiple qPCR reaction, any one in two appraisal mark objects is the positive for positive findings and two
When as a result, which is judged to positive sample.
Further, sample is studied: totally 1284 parts of Urogenital Specimens for leaving and taking sexually transmitted disease patient at random, including
Swab and totally 1048 parts of vaginal swab in 236 parts of male urethra swab, women uterine neck are evaluated for method.
The beneficial effects of the present invention are:
(1) Monitoring lower-cut expands the range of linearity and reaches 1.0 × 102-1.0 × 108copies/ down to 2copies/PCR
ML, diagnostic sensitivity and specificity respectively 100.0% (134/134) to chlamydia trachomatis, 99.3% (1142/1150),
From sample treatment to report result≤2.0 hour；
(2) this method patent simplicity, quick, high sensitivity, specificity are good, and examining for chlamydia trachomatis infection not only can be improved
Cutting capacity can also be achieved quick detection, for precisely treatment gains time as early as possible.
[Detailed description of the invention]
Fig. 1 is crypticplasmid primer of the present invention, probe in detecting chlamydia trachomatis gradient template result figure；
Fig. 2 is 23S rRNA primer of the present invention, probe in detecting chlamydia trachomatis gradient template result figure；
Fig. 3 is the CT Distribution value figure of 134 multiple qPCR positive samples of the invention；
[specific embodiment]
With reference to the accompanying drawing and specific embodiment is described further the present invention:
As shown in Figure 1, Figure 2, Figure 3 shows, a kind of quickly detection dual qPCR method of chlamydia trachomatis, includes the following steps:
(1) early-stage preparations program:
Research sample: leaving and taking the Urogenital Specimens several pieces of sexually transmitted disease patient at random, including male urethra is wiped
Swab and vaginal swab several pieces in sub- several pieces, women uterine neck are evaluated for method.
Instrument and reagent:
1. instrument for extracting nucleic acid (ExPure20) and its mating paramagnetic particle method extraction purification reagent；
2. fluorescence quantitative PCR instrument (LightCycler480)；Multiple fluorescence quantitative PCR system (PlatinumTM
Multiplex PCR Master Mix)；
3. PCR product is sequenced；
(2) nucleic acid extraction and purifying:
1. original sample nucleic acid extraction: carrying out nucleic acid extraction with genital tract sample nucleic acid extracts kit, extraction operation is tight
Lattice are carried out according to instrument and matched reagent SOP；
2. nucleic acid-templated sucking after extraction purification is managed without DNA enzymatic EP, be stored in -86 DEG C freeze it is spare.
(3) primer/probe design, synthesis, label, from ncbi database, download the chlamydia trachomatis blood announced respectively
Clear type D-K bacterial strain cryptic plasmid and 23S rRNA gene order, carry out sequence alignment with DNAMAN, with 5 software of Primer, keep away
After opening region of variability or catastrophe point, primer and probe synthesis and the label table of each gene are separately designed are as follows:
(4) qPCR amplification and result judgement:
1. multiple qPCR system: 2 × PlatinumTM25.0 μ L of Multiplex PCR Mix, 50 μM of positive anti-primer are each
Each 0.5 μ L of 0.3 μ L, 10 μM of probe, 20.0 μ L of template, moisturizing to 50.0 μ L of final volume；
2. Amplification: 95 DEG C of 2.5min+ (95 DEG C of 10s+60 DEG C of 30s) × 45 select the channel 510nm and 580nm in 60
DEG C detection fluorescence；
3. qualification result judges: the amplification curve of appraisal mark object is in serpentine, and CT value < 37 is judged to the positive；Amplification curve
In serpentine, 37≤CT value < 40 then carries out repeating to detect twice again, if amplification curve remains as serpentine, CT value still < 40,
Also it is judged to the positive；Otherwise it is judged to feminine gender；
4., should when any one or two in two appraisal mark objects are positive findings in multiple qPCR reaction
Sample is judged to positive sample；
(5) Monitoring lower-cut and amplification range of linearity test:
1. the high concentration chlamydia trachomatis nucleic acid template after concentration is carried out 10 times of gradient dilutions with ultrapure water, concentration is made
For 1.0 × 102-1.0 × 108copies/mL gradient concentration template, it to be used for PCR amplification；The minimum addition template of sun can be sentenced
Number is used as Monitoring lower-cut；Using the good minimum and maximum concentration of linear relationship as expanding the range of linearity；
(6) diagnostic sensitivity, specificity calculate:
1. original sample qualification result judges: multiple qPCR method and substance commercial kit are positive, then are judged to kidney-Yang
Property；Multiple qPCR method is positive, and substance commercial kit is negative, is sequenced through an amplified production generation, with mark strain homology >=90%；
Also true positives are judged to；Otherwise, it is judged to false positive；
2. multiple qPCR method is negative, substance commercial kit is negative, then is judged to true negative；Multiple qPCR method is negative, single
Weight commercial kit is positive, is sequenced through amplified production, with mark strain homology >=90%, is judged to false negative；Otherwise, it is also judged to true
It is negative；
3. diagnostic sensitivity=true positives number ÷ (true positives number+false negative number) × 100%；Specificity=true negative
Number ÷ (true negative number+false positive number) × 100%；
(7) statistical method: carrying out student's t check analysis with SPSS to all data, and P < 0.05 is to have statistical difference, and P <
0.01 is to have significant statistical difference.
Preferably, the sequence of chlamydia trachomatis cryptic plasmid and 23S rRNA genetic test primer and probe with
Mark table are as follows:
Preferably, the nt:1755-1880 segment of Simultaneous detection of Chlamydia trachomatis cryptic plasmid and 23S rRNA gene
Nt:51-169 segment, the primer and probe are determined after China's Distribution strain screening, and region of variability or catastrophe point are avoided
It designs.
Preferably, the dual qPCR reagent of chlamydia trachomatis is quickly detected:
(1) multiple qPCR system: 2 × PlatinumTM25.0 μ L of Multiplex PCR Mix, 50 μM of positive anti-primer
Each 0.5 μ L of each 0.3 μ L, 10 μM of probe, 20.0 μ L of template, moisturizing to 50.0 μ L of final volume；
(2) Amplification: 95 DEG C of 2.5min+ (95 DEG C of 10s+60 DEG C of 30s) × 45 select the channel 510nm and 580nm in 60
DEG C detection fluorescence.
Preferably, the dual qPCR method of chlamydia trachomatis, qualification result judgment method are quickly detected are as follows:
(2) amplification curve of appraisal mark object is in serpentine, and CT value < 37 is judged to the positive；Amplification curve be in serpentine, 37≤
CT value < 40 then carries out repeating to detect twice again, if amplification curve remains as serpentine, CT value still < 40 is also judged to the positive；It is no
Then it is judged to feminine gender；
In multiple qPCR reaction, any one in two appraisal mark objects is the positive for positive findings and two
When as a result, which is judged to positive sample.
Preferably, sample is studied: totally 1284 parts of Urogenital Specimens for leaving and taking sexually transmitted disease patient at random, including male
Property 236 parts of urethral swab, swab and totally 1048 parts of vaginal swab in women uterine neck, evaluated for method.
As point each in Fig. 3 represents a sample；When CT value=45 indicate there is no curve when detecting the segment of the sample
Take-off, the segment testing result are feminine gender；In multiple qPCR reaction, any one in two appraisal mark objects is positive knot
When fruit and two are positive findings, which is judged to positive sample.
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out change and modification appropriate.Therefore, the invention is not limited to the specific embodiments disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In use some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Claims (4)
1. the quickly detection dual qPCR primer and probe of chlamydia trachomatis, using Real-Time Fluorescent Quantitative PCR Technique
(Quantitative Real-time PCR, qPCR), it is characterised in that: chlamydia trachomatis cryptic plasmid and 23S
The sequence of rRNA genetic test primer and probe and label table are as follows:
2. the dual qPCR method of quick detection chlamydia trachomatis according to claim 1, it is characterised in that: while detecting sand
The nt:1755-1880 segment of chlamydia oculogenitale cryptic plasmid and the nt:51-169 segment of 23S rRNA gene, the primer and spy
Needle is determined after China's Distribution strain screening, avoids region of variability or catastrophe point designs.
3. the dual qPCR reagent of quick detection chlamydia trachomatis according to claim 1, it is characterised in that:
(1) multiple qPCR system: 2 × PlatinumTM25.0 μ L of Multiplex PCR Mix, 50 μM of positive anti-primer each 0.3
Each 0.5 μ L of μ L, 10 μM of probe, 20.0 μ L of template, moisturizing to 50.0 μ L of final volume；
(2) Amplification: 95 DEG C of 2.5min+ (95 DEG C of 10s+60 DEG C of 30s) × 45 select the channel 510nm and 580nm to visit in 60 DEG C
Survey fluorescence.
4. the dual qPCR method of quick detection chlamydia trachomatis according to claim 1, which is characterized in that qualification result is sentenced
Disconnected method are as follows:
(1) amplification curve of appraisal mark object is in serpentine, and CT value < 37 is judged to the positive；Amplification curve is in serpentine, 37≤CT value
< 40 then carries out repeating to detect twice again, if amplification curve remains as serpentine, CT value still < 40 is also judged to the positive；Otherwise sentence
For feminine gender；
(2) in multiple qPCR reaction, any one in two appraisal mark objects is the positive for positive findings and two
When as a result, which is judged to positive sample.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041864A (en) * | 2007-03-06 | 2007-09-26 | 扬子江药业集团北京海燕药业有限公司 | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method |
| CN101363041A (en) * | 2008-05-04 | 2009-02-11 | 卫生部北京医院 | Quality control substance for detecting chlamydi trachomatis |
| CN102094076A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for quantitatively detecting chlamydia trachomatis infection |
| CN109321636A (en) * | 2018-10-09 | 2019-02-12 | 中国农业大学 | A kind of chip and application for the detection of Chlamydia species specificity |
-
2019
- 2019-03-01 CN CN201910153964.2A patent/CN109750115A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101041864A (en) * | 2007-03-06 | 2007-09-26 | 扬子江药业集团北京海燕药业有限公司 | Chlamydi trachomatis and Neisseria gonorrhoeae dual real-time fluorescence PCR detection method |
| CN101363041A (en) * | 2008-05-04 | 2009-02-11 | 卫生部北京医院 | Quality control substance for detecting chlamydi trachomatis |
| CN102094076A (en) * | 2009-12-11 | 2011-06-15 | 上海裕隆临床检验中心有限公司 | Fluorescent polymerase chain reaction (PCR) kit for quantitatively detecting chlamydia trachomatis infection |
| CN109321636A (en) * | 2018-10-09 | 2019-02-12 | 中国农业大学 | A kind of chip and application for the detection of Chlamydia species specificity |
Non-Patent Citations (1)
| Title |
|---|
| MA等: "Rapid and accurate diagnosis of Chlamydia trachomatis in the urogenital tract by a dual-gene multiplex qPCR method", 《JOURNAL OF MEDICAL MICROBIOLOGY》, vol. 68 * |
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