CN108546746A - Based on RNA target mark SAT chlamydia trachomatis infection molecular biology for detection - Google Patents
Based on RNA target mark SAT chlamydia trachomatis infection molecular biology for detection Download PDFInfo
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- CN108546746A CN108546746A CN201810454077.4A CN201810454077A CN108546746A CN 108546746 A CN108546746 A CN 108546746A CN 201810454077 A CN201810454077 A CN 201810454077A CN 108546746 A CN108546746 A CN 108546746A
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Abstract
The present invention provides a kind of chlamydia trachomatis infection molecular biology for detection of the SAT based on RNA target mark, include the following steps：Step 1, using specific target capture technique, paramagnetic particle method extracts pure cell target nucleic acid, i.e. RNA as target, including：(1 1) manual extraction target nucleic acids specificity；(1 2) pure cell target nucleic acid, i.e., RNA is as target；Step 2 obtains the testing result of chlamydia trachomatis infection using RNA real-time fluorescence constant-temperature amplification detection techniques, including：(2 1) M MLV reverse transcriptase generates a DNA copy of target nucleic acids RNA；(2 2) T7 RNA polymerases generate multiple RNA copies from DNA copy；The RNA of optimization probe and extraction with fluorescent marker is copied specific bond by (2 3), is captured by detecting instrument to generate fluorescence signal, the testing result of chlamydia trachomatis infection is obtained according to the time of occurrence of signal and intensity；Step 3 judges the testing result in conjunction with positive control and negative control.
Description
Technical field
The present invention relates to a kind of molecular Biological Detection of bacterium infection and identification technology field more particularly to trachoma clothing are former
The method of the molecular Biological Detection and identification of body-sensing dye.
Background technology
Chlamydia be it is a kind of can by bacterial filter, have unique growth cycle, stringent cytozoicus prokaryotic cell type
Microorganism, including four kinds of chlamydia trachomatis, chlamydia psittaci, chlamydia pneumoniae and domestic animal Chlamydia.Chlamydia trachomatis
(chlamydial tyachomatis, CT) be trachoma, non-gonococcal urethritis, cervicitis, pelvic infecton, endometritis, new
The pathogen of a variety of diseases such as raw youngster's inclusion conjunctivitis, infant pneumonia, is the disease of most popular sexually transmitted disease in the world
Substance.The laboratory diagnosis of CT infection includes mainly cell biology method (direct microscopy, be separately cultured), immunological method
(direct fluorescence antibody detection, enzyme-linked immunosorbent assay) and molecular biology method.
Molecular biology method prior art generally use is real-time fluorescence nucleic acid constant-temperature amplification detection technique
(Simultaneous Amplification and Testing, abbreviation SAT), the technology are to expand the nucleic acid constant-temperature of a new generation
A kind of novel nucleic acids detection technique that increasing technology and real-time fluorescence detection technique are combined.Have compared with traditional detection method
High sensitivity, high specific, low stain and stable reaction.
About the detection technique of external pathogen, American-European disease control department and WHO recommend, and Molecular diagnostics are detected as CT
A kind of means, be presently the most sensitive and accurate method.Moreover, only Molecular diagnostics can detect urine, because of sampling
It is convenient, it is highly suitable for the universal screening of CT.In the U.S., isothermal amplification technology (TMA) becomes mainstream with 64% absolute predominance
Detection technique.Foreign study shows that the technology is not influenced by specimen sampling, and sampling is convenient, high sensitivity, and specificity is good,
It may be simultaneously used for the monitoring of medicining condition and sentence more.For hospital, its whole treatment level can be improved, to patient
For, then it avoids and goes to a doctor repeatedly, diagnosis and treatment cost can be reduced.Overseas market is the amplification of the transcriptive intermediate of Gen-Probe companies
(TMA), expand and detect the rRNA of CT.Advantage is that have a large amount of rRNA in each infection cell, and operating procedure is easy, only
It is a thermostatic process, does not need the amplification instrument of transformation temperature.Since detection method is end point determination, it is easy to cause environment dirt
Dye, the country there is no the report being detected using the method.The current country is mainly colloidal gold and PCR to the detection of venereal disease CT.But
Culture is not easy, bad, the pollution problem of PCR of colloidal gold testing result, has also caused the clinical expectation detected to RNA.Doctor
Institute's routine CT samples are urethral swab and Cervical scrapes (being partly vaginal swab), need the medical worker of profession to carry out sample and adopt
Collection, for male patient, collecting sample painful.CT-SAT constant-temperature amplification detection kits are that unique SFDA that obtains ratifies
, it can be used for detecting the diagnostic reagent of urine specimen, high sensitivity, specificity is good, and gene-amplification product is RNA, can avoid DNA
Caused contaminated clinical, however its sampling still falls within intrusion type, patient is more painful.
RNA detection techniques can not only detect urethra and Cervical scrapes, but also can detect urine specimen.It is with cultivation
Goldstandard is the CT for expanding goldstandard using SAT detection men and women's genital tract samples, the sensitivity of swab specimen with PCR methods
97.0%, specificity 99.1%, accuracy 98.6%；The sensitivity 95.8% of first void urine sample, specificity 99.1%,
Accuracy 98.3%.The selection of urine specimen can mitigate patient suffering, reduce medical worker's workload.Due to RNA detection of nucleic acids
The high sensitivity and specificity of technology may be used this method conventional detection and answer in the laboratory for having molecular biology condition
With.Sanger sequencings are the international goldstandards of current all genetic tests！Sanger sequencings are the states of current all genetic tests
Border goldstandard, it includes quantitative fluorescent PCR Taqman sonde methods, regular-PCR method, chip method, two generation PCR sequencing PCRs, mass spectrography etc. to be
The goldstandard of method.Scientific research field delivers genetic test related article, it is necessary to have sanger sequence verification data to support.
Sanger sequencings why be current genetic test international goldstandard, be because of the unusual science of sanger sequencing principles, process
Very careful, real result is visual, and accuracy rate is very high, reaches 99.999%.Library need not be built, belongs to direct Sequencing, directly
Read as a result, continuously read data (not being a point), conclusion need not be derived, the bright and clear data supporting of process is abundant.This skill
Although art is to have passed through 38 years, application is still extensive, or the main force being sequenced, and a lot of other methods that detect cannot substitute.
However, how to extract pure RNA as target is also current a great problem.
The detection method of the prior art is subjected to a comparison, referring to table 1, it can be found that prior art detection method is bright
Aobvious defect is：It operates more complex, need to have certain experiences that can just carry out detection, and time-consuming, manual operations, it is more complex.
1 each Comparison between detecting methods table of table
Invention content
In order to overcome the disadvantages described above in prior art detection method, this application provides the sand of the SAT based on RNA target mark
Ocular Chlamydia infection molecular biology for detection, inventive concept are：RNA is obtained by specific target capture technique,
And organically combine the advantages of RNA target mark and SAT technologies with the combination of real-time fluorescence constant-temperature amplification detection technique (SAT), certainly
Technical scheme of the present invention is not the simple superposition of two kinds of technologies application, but by specific target capture technique, also may be used
Pure bacterium target nucleic acid (RNA) is obtained as the most important base of accurate detection chlamydia trachomatis infection using referred to as paramagnetic particle method
Plinth；Real-time fluorescence nucleic acid constant-temperature amplification detection technique is come using M-MLV reverse transcriptase, T7RNA polymerases and optimization probe technique
It realizes simultaneously, it is more complex to overcome operation, there need to be certain experiences that can just carry out detection, and time-consuming, manual operations, it is more multiple
Miscellaneous defect.
A kind of chlamydia trachomatis infection molecular biology for detection of the SAT based on RNA target mark, includes the following steps：
Step 1, using specific target capture technique, i.e. paramagnetic particle method extracts pure cell target nucleic acid, i.e. RNA conducts
Target；
Step 2 obtains the testing result of chlamydia trachomatis infection, institute using RNA real-time fluorescence constant-temperature amplification detection techniques
RNA real-time fluorescence constant-temperature amplification detection techniques are stated using reverse transcriptase, RNA polymerases and optimization probe while being realized；
Step 3 judges the testing result in conjunction with positive control and negative control.
Preferably, the step 1 includes：
(1-1) manual extraction target nucleic acids specificity：It is included in bead particulates surface markers oligo (dT), in the section
The trapping nucleic acids segment of specificity of the connection with one section of oligo (dA) on oligo (dT), when capture probe and target nucleic acids knot
After conjunction, with magnet adsorption magnetic bead, you can extraction target nucleic acids specificity；
(1-2) obtains pure bacterium target nucleic acid, i.e., RNA is as target：During being included in nucleic acid extraction, pathogen
The nucleic acid released and the magnetic-particle specific bond in nucleic acid extraction liquid are cracked, cleaning magnetic-particle obtains pure bacterium target
Nucleic acid RNA to greatest extent gets rid of the impurity in sample, ensures specificity when reaction.
Preferably, the step 2 includes：
(2-1) reverse transcriptase is M-MLV reverse transcriptase, generates a DNA copy of target nucleic acids RNA；
(2-2) RNA polymerases are T7RNA polymerases, and multiple RNA copies are generated from DNA copy；
The RNA of optimization probe and step 1 extraction with fluorescent marker is copied specific bond by (2-3), to produce
Raw fluorescence, the fluorescence signal is captured by detecting instrument, according to the time of occurrence and intensity of the fluorescence signal obtained in real time
Obtain the testing result of chlamydia trachomatis infection.
Preferably, the step 3 combination positive control and negative control carry out judgement to the testing result and include：It answers
Laboratory Quality Control is carried out with the control of critical weakly positive, set positive control, critical weakly positive compare and the dt of negative control
It should meet：Positive control dt≤critical weakly positive compares dt≤35, and negative control dt is without numerical value, or is 40, and otherwise experiment regards
It is invalid, re-starts, wherein dt indicates that sample curve and the abscissa of threshold curve intersection point are read.
Preferably, step (1-1) the manual extraction target specificity and step (1-2) obtain pure bacterium target nucleus
Acid uses Beads enrichment device, electric suction apparatus, dry type thermostat and continuous micro sample injector to complete.
Preferably, step (1-1) the manual extraction target specificity and step (1-2) obtain pure bacterium target nucleus
Acid can also complete all extraction process by MagX Full automatic instrument for extracting nucleic acid, and the nucleic acid extracted is in fluorescent PCR instrument
Augmentation detection is carried out on device.
SAT technologies after new improvement draw the advantage of various technologies, are expanded, are gone forward side by side as template using pure RNA
Row detection in real time, and sampled using non-intrusion type sample, it is effectively saved medical resource, and offer convenience to patient, detected
It is at low cost, more patients can be indebted to, as newest diagnostic techniques, there is quick, sensitive, special, easy, greatly reduction leakage
Inspection, false drop rate.
According to the following detailed description of specific embodiments of the present invention in conjunction with the accompanying drawings, those skilled in the art will be brighter
The above and other objects, advantages and features of the present invention.
Description of the drawings
Some specific embodiments that the invention will be described in detail by way of example and not limitation with reference to the accompanying drawings hereinafter.
Identical reference numeral denotes same or similar component or part in attached drawing.It should be appreciated by those skilled in the art that these
What attached drawing was not necessarily drawn to scale.The target and feature of the present invention will be apparent from view of following description taken together with the accompanying drawings,
In attached drawing：
Fig. 1 is to use specific target capture technique, i.e. paramagnetic particle method to extract pure cell according to the embodiment of the present invention
The schematic diagram of target nucleic acid, i.e. RNA as target；
Fig. 2 is to obtain chlamydia trachomatis using RNA real-time fluorescence constant-temperature amplification detection techniques according to the embodiment of the present invention
The schematic diagram of the testing result of infection.
Specific implementation mode
The present embodiment is directed to a kind of chlamydia trachomatis infection molecular biology for detection of the SAT based on RNA target mark, leads to
Cross specific target capture technique and obtain pure RNA, and and fluorescence constant-temperature amplification detection technique (SAT) combination by RNA target mark
It is organically combined with the advantages of SAT technologies, certain technical scheme of the present invention is not the simple superposition of two kinds of technologies, but passes through
Specific target capture technique is referred to as paramagnetic particle method and obtains pure bacterium target nucleic acid (RNA) as accurate detection trachoma
The most important basis of choamydiae infection；Real-time fluorescence nucleic acid constant-temperature amplification detection technique uses M-MLV reverse transcriptase, T7RNA
Polymerase and optimization probe technique are come while being realized, include the following steps：
Referring to Fig. 1, step 1, using specific target capture technique, i.e. paramagnetic particle method extracts pure cell target nucleic acid, i.e.,
RNA uses Beads enrichment device, electric suction apparatus, dry type thermostat and continuous micro sample injector to complete as target, certainly also
All extraction process can be completed by MagX Full automatic instrument for extracting nucleic acid, the nucleic acid extracted is on fluorescent PCR instrument
Augmentation detection is carried out, concrete operations include：
(1-1) manual extraction target nucleic acids specificity：It is included in bead particulates surface markers oligo (dT), in the section
The trapping nucleic acids segment of specificity of the connection with one section of oligo (dA) on oligo (dT), when capture probe and target nucleic acids knot
After conjunction, with magnet adsorption magnetic bead, you can extraction target nucleic acids specificity；
(1-2) obtains pure bacterium target nucleic acid, i.e., RNA is as target：During being included in nucleic acid extraction, pathogen
The nucleic acid released and the magnetic-particle specific bond in nucleic acid extraction liquid are cracked, cleaning magnetic-particle obtains pure bacterium target
Nucleic acid RNA to greatest extent gets rid of the impurity in sample, ensures specificity when reaction；
Referring to Fig. 2, step 2 obtains the inspection of chlamydia trachomatis infection using RNA real-time fluorescence constant-temperature amplification detection techniques
It surveys as a result, the RNA real-time fluorescences constant-temperature amplification detection technique uses reverse transcriptase, RNA polymerases and optimization probe while reality
It is existing；Including：
(2-1) reverse transcriptase is M-MLV reverse transcriptase, generates a DNA copy of target nucleic acids RNA；
(2-2) RNA polymerases are T7RNA polymerases, and multiple RNA copies are generated from DNA copy；
The RNA of optimization probe and step 1 extraction with fluorescent marker is copied specific bond by (2-3), to produce
Raw fluorescence, the fluorescence signal is captured by detecting instrument, according to the time of occurrence and intensity of the fluorescence signal obtained in real time
Obtain the testing result of chlamydia trachomatis infection；
Step 3 judges the testing result in conjunction with positive control and negative control, including：Using critical weak sun
Property control carry out laboratory Quality Control, set positive control, the control of critical weakly positive and the dt of negative control should meet：It is positive
It compares dt≤critical weakly positive and compares dt≤35, negative control dt is without numerical value, or is 40, and otherwise experiment is considered as invalid, again
It carries out, wherein dt indicates that sample curve and the abscissa of threshold curve intersection point are read.
Table 2 is the place and equipment situation that the present embodiment is implemented.
Table 2
SAT technologies after new improvement draw the advantage of various technologies, are expanded, are gone forward side by side as template using pure RNA
Row detection in real time has quick, sensitive, special, easy as newest diagnostic techniques, greatly reduces missing inspection, false drop rate.
It, will not be by these embodiments although the present invention is described by reference to specific illustrative embodiment
Restriction and only limited by accessory claim.It should be understood by those skilled in the art that can be without departing from the present invention's
The embodiment of the present invention can be modified and be changed in the case of protection domain and spirit.
Claims (6)
1. a kind of chlamydia trachomatis infection molecular biology for detection of the SAT based on RNA target mark, it is characterised in that including such as
Lower step：
Step 1, using specific target capture technique, i.e. paramagnetic particle method extracts pure cell target nucleic acid, i.e., RNA is as target；
Step 2 obtains the testing result of chlamydia trachomatis infection using RNA real-time fluorescence constant-temperature amplification detection techniques, described
RNA real-time fluorescence constant-temperature amplification detection techniques are realized simultaneously using reverse transcriptase, RNA polymerases and optimization probe；
Step 3 judges the testing result in conjunction with positive control and negative control.
2. the chlamydia trachomatis infection molecular Biological Detection side of SAT based on RNA target mark according to claim 1 a kind of
Method, it is characterised in that the step 1 includes：
(1-1) manual extraction target nucleic acids specificity：It is included in bead particulates surface markers oligo (dT), in this section of oligo
(dT) the trapping nucleic acids segment of specificity of the connection with one section of oligo (dA) on, after capture probe is combined with target nucleic acids,
With magnet adsorption magnetic bead, you can extraction target nucleic acids specificity；
(1-2) obtains pure bacterium target nucleic acid, i.e., RNA is as target：During being included in nucleic acid extraction, pathogen cracking
The nucleic acid released and the magnetic-particle specific bond in nucleic acid extraction liquid, cleaning magnetic-particle obtain pure bacterium target nucleic acid
RNA to greatest extent gets rid of the impurity in sample, ensures specificity when reaction.
3. the chlamydia trachomatis infection molecular Biological Detection side of SAT based on RNA target mark according to claim 1 a kind of
Method, it is characterised in that the step 2 includes：
(2-1) reverse transcriptase is M-MLV reverse transcriptase, generates a DNA copy of target nucleic acids RNA；
(2-2) RNA polymerases are T7RNA polymerases, and multiple RNA copies are generated from DNA copy；
The RNA of optimization probe and step 1 extraction with fluorescent marker is copied specific bond by (2-3), glimmering to generate
Light, the fluorescence signal are captured by detecting instrument, are obtained according to the time of occurrence of the fluorescence signal obtained in real time and intensity
The testing result of chlamydia trachomatis infection.
4. the chlamydia trachomatis infection molecular Biological Detection side of SAT based on RNA target mark according to claim 1 a kind of
Method, it is characterised in that the step 3 combination positive control and negative control carry out judgement to the testing result and include：Using
Critical weakly positive control carries out laboratory Quality Control, and the dt of set positive control, the control of critical weakly positive and negative control is answered
Meet：Positive control dt≤critical weakly positive compares dt≤35, and negative control dt is without numerical value, or is 40, and otherwise experiment is considered as
In vain, it re-starts, wherein dt indicates that sample curve and the abscissa of threshold curve intersection point are read.
5. the chlamydia trachomatis infection molecular Biological Detection side of SAT based on RNA target mark according to claim 2 a kind of
Method, it is characterised in that step (1-1) the manual extraction target specificity and step (1-2) obtain pure bacterium target nucleic acid
It is completed using to Beads enrichment device, electric suction apparatus, dry type thermostat and continuous micro sample injector.
6. the chlamydia trachomatis infection molecular Biological Detection side of SAT based on RNA target mark according to claim 2 a kind of
Method, it is characterised in that step (1-1) the manual extraction target specificity and step (1-2) obtain pure bacterium target nucleic acid
All extraction process can also be completed by MagX Full automatic instrument for extracting nucleic acid, the nucleic acid extracted is in fluorescent PCR instrument
Upper carry out augmentation detection.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333564A (en) * | 2007-06-27 | 2008-12-31 | 上海仁度生物科技有限公司 | Method for extracting and purifying target nucleic acid and use thereof |
| CN101509041A (en) * | 2009-03-20 | 2009-08-19 | 上海仁度生物科技有限公司 | Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333564A (en) * | 2007-06-27 | 2008-12-31 | 上海仁度生物科技有限公司 | Method for extracting and purifying target nucleic acid and use thereof |
| CN101509041A (en) * | 2009-03-20 | 2009-08-19 | 上海仁度生物科技有限公司 | Chlamydia trachomatis nucleic acid detection kit for constant-temperature amplification by using RNA |
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