CN102888464A - Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof - Google Patents
Quintuple fluorescent PCR (polymerase chain reaction) quick and hypersensitive detection kit and application thereof Download PDFInfo
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Abstract
The invention relates to a real-time PCR (polymerase chain reaction) method for quintuply detecting target nucleic acid in a nucleic acid extracting solution in a single PCR reaction vessel, which is used for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium in a sample.
Description
Technical field
The invention belongs to the nucleic acid detection technique field, particularly, the present invention relates to can be simultaneously to five kinds of Multiplex real-time PCR methods that the infective pathogen body detects such as chlamydia trachomatis, Diplococcus gonorrhoeae, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitaliums, its quick, super quick and one the step finish.In addition, the invention still further relates to reagent related in the above-mentioned PCR method, as mutually irrelevant around primer, probe etc., and be used for the detection kit of aforesaid method and the Application and preparation of corresponding detection kit etc.
Background technology
Diplococcus gonorrhoeae (NG), chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), mycoplasma hominis (MH), mycoplasma genitalium (MG) are 5 kinds of main pathogens that cause urogenital infections, can cause urethritis, prostatitis, cervicitis, salpingitis even infertile etc.Wherein, Diplococcus gonorrhoeae (Niesseria gonorrhoeae, NG) is the pathogenic agent that causes gonorrhoea, and gonorrhoea is modal disease in the sexually transmitted disease (STD) always, can cause that serious urogenital tract disease, especially female patient often cause inflammatory pelvic disease and fertility; Chlamydia trachomatis (Chlamydia trachomatis, CT) also is one of pathogenic agent of important sexually transmitted disease (STD), can cause vagina, urethra and up genital tract infection, also can cause pelvic inflammatory disease, uterine tube damage; Ureaplasma urealyticum (Ureaplasma urealyticum, UU) is the main pathogens that causes inflammation of genitourinary tracts (NGU) and male and female infertility, also is the common disease substance that spreads through sex intercourse, and is common urogenital parasite; Mycoplasma hominis (Mycoplasma hominis, MH) is a kind of common microorganism of adult's urogenital tract, and is relevant with some gynaecology, andropathy, also infects with the newborn respiration road, central nervous system infection is relevant; Mycoplasma genitalium (Mycoplasma genitalium, MG) can cause nongonococcal urethritis (NGU), prostatitis, cervicitis, endometritis, appendagitis and infertile, may in HIV infects, play an important role simultaneously, be called as " AIDS be correlated with mycoplasma ".
The U.S. is since nineteen ninety-five is carried out reproductive tract choamydiae infection case report work, and its sickness rate is very high, more than 180/ ten thousand; The asab sickness rate is higher, and is comparatively perfect such as Zimbabwe's sanitation system, the whole nation more than 1,000 ten thousand populations, and the VC of 1 annual report just reaches 800,000, accounts for total population nearly 10%.And deepening continuously along with China's reform and opening-up, social economy's development, movement of population is constantly strengthened, the infection of venereal disease the trend that rises arranged year by year, China's gonorrhoea and syphilis sickness rate are higher than developed country at present, be lower than African country, and the sickness rate of China's venereal disease still keeps rapid growth, 1991-2000 whole nation venereal disease is flat to increase by 19.30%, especially syphilis, genital herpes and NGU increasing degree are more than 40%, and venereal disease and acquired immune deficiency syndrome (AIDS) is popular closely related, the popular propagation that will promote acquired immune deficiency syndrome (AIDS) of venereal disease.
Now to the detection of Diplococcus gonorrhoeae, Ureaplasma urealyticum, mycoplasma hominis, mycoplasma genitalium mainly take culture method as main; Chlamydia trachomatis carries out immunology detection mainly for its antigen.Culture method detects length consuming time, generally needs just can obtain the result through 48-72 hour; Sensitivity is low, is subjected to gather, transports, preservation etc. affects greatlyr, false negative result easily occurs; Operate loaded down with trivial detailsly, cause the as a result discrimination standard of various operations to be difficult to unified, the technology and equipment that needs requires high.Immunological method is compared culture method significant progress, but because the cross reaction that exists between the reproductive tract microorganism so that the specificity of the method is affected, false positive reaction easily occurs, brings the possibility of erroneous judgement to treatment.
In recent years also there is report to utilize polymerase chain reaction (PCR) to implement detection to pathogenic agent.PCR is one of technology the most frequently used in the current detection of nucleic acids, wherein in real time (Real Time) round pcr has adopted the mark that can detect in real time such as fluorescent mark etc., at aspects such as detection of nucleic acids, clinical diagnosis and molecular biology researches, can obtain rapidly the result.Conventional PCR in real time detects, such as Chinese patent CN1768151A, CN101189505A, CN101273262A, CN101302473A etc., only for detection of single target nucleic acid; Chinese patent CN101624629A discloses the real-time PCR method of target nucleic acid in (in single PCR reaction vessel) Multiple detection sample, execution speed is faster, more integration, but it is only for detection of three kinds of target nucleic acids such as HBV, HCV and HIV, there is not the more practice of Multiple detection, for example not about how designing the prompting of non-interfering primer pair and probe, more do not point out adjusting concentration and probe concentration.
Yet, detect for more multiple PCR in real time, especially for Diplococcus gonorrhoeae (NG), chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), mycoplasma hominis (MH), these five kinds of pathogenic agent (wherein the homology of three mycoplasma species is higher) of mycoplasma genitalium (MG), by present known primer pair and probe sequence method of design (comprising existing bioinformatics software), can't design this five heavy non-interfering primer pair and probe, design will cause interfering with each other and so that the distortion of fluoroscopic examination curve.For this reason, the inventor is through painstaking efforts and relied on some fortune, for these five kinds of pathogenic agent, the degree of interfering with each other acceptable primer pair and probe in PCR in real time detects have been designed, more surprisingly, adjust in concentration and probe concentration, strengthened they detection curve can both sharpness and resolution (curve is separated from each other), so that detection sensitivity has reached super quick level, and can not use internal reference.In addition, the present invention is optimized for the method for extracting nucleic acid of mentioned microorganism.
Summary of the invention
The problem that will solve of the present invention is to provide five new heavy real-time PCR methods, be used for five re-detection sample Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium, by interfering with each other degree acceptable primer pair and probe in PCR in real time detects, and the optional process of optimizing concentration and probe concentration and nucleic acid extraction, tell simultaneously the target nucleic acid that whether exists in the sample from mentioned microorganism high specific, highly sensitive and high duplication.In addition, the invention still further relates to detection kit that aforesaid method is provided and application etc.
Particularly, in first aspect, the invention provides in single PCR reaction vessel the real-time PCR method of target nucleic acid in the five re-detection nucleic acid extraction liquid, described target nucleic acid is from Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium, it comprises
(1) mixed nucleus acid extraction liquid, archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes in described single PCR reaction vessel, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises
NG-F:ACCGTAACGTCTCTAAGTC，
NG-R:GCAAGATTTCCGATTTGGC，
CT-F:TGTCCATATCTTTGATACGAT，
CT-R:CTCTGATATTTGAAGACTCTACA，
UU-F:CTGCTCGTGAAGTATTACA，
UU-R:GTACCATCTGGGAAAGTAC，
MH-F:TGTGGTTTAATTTGAAGATACAT，
MH-R:AGTCCTCAACTTAATGTTAGA，
MG-F:AGGCAGTAATGTCAATCACA, and
MG-R:GGCTTTATCATTGGCAAACG，
Target nucleic acid probes comprises:
NG-P:CAGACATCACGCACCGAAGCAT，
CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA，
UU-P:ACCAACCATTGTATCTACACCTTCCATA，
MH-P:CCACTCTTGACATCCTTCGCATA, and
MG-P:CCATTCGCATCCAACTTCACCTCT；
(2) carry out the PCR reaction, and detect in real time the fluorescence of different wave length; With,
(3) the Ct value that calculates according to the fluoroscopic examination result has judged whether that target nucleic acid is present in the sample.
Can carry out direct-detection to nucleic acid extraction liquid, but detect again after preferably sample being processed rear acquisition nucleic acid extraction liquid.So the preferred nucleic acid extracting solution is to extract the nucleic acid in the sample and the extracting solution that obtains, adopts in this way the magnetic bead extraction method to extract.Nonspecific magnetic bead extraction method adopts the surface to scribble the paramagnetic particle of non-specific nucleic acid absorption class material, low pH (as, pH value 5 ~ 7) nucleic acid can be adsorbed onto the paramagnetic particle surface and in the situation of high salt concentration, after carrying out the magnetic separation and fully washing, high pH (as, pH value 8 ~ 9), nucleic acid can be eluted under the condition of low salt concn, thus enrichment the sample of nucleic acid (such as target nucleic acid) can be used for the PCR test; Can also adopt in addition the magnetic bead extraction method of specific adsorption (such as hybridization absorption and immunosorption).These treating processess are known to those skilled in the art, also can be referring to Zheng Xiufen etc., template DNA magnetic bead extraction method. the Chinese law medical journal, 18(3): 107-108; Chinese patent 200610030229.5,200710118802.2 and 201110105181.0 etc.
The magnetic bead extraction method that the preferred inventor of employing further optimizes, it is more reliable for the nucleic acid that extracts simultaneously in Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and the mycoplasma genitalium, and step is also convenient.The preferred extraction is subjected to the process of the nucleic acid in the test product to be: (the preferred nucleic acid extraction liquid comprises guanidinium isothiocyanate to add the nucleic acid extraction liquid to sample, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl)), add magnetic bead after the insulation, after mixing, after applying magnetic field, discard wherein liquid, then washing (preferred washed twice, more preferably wherein for the first time wash used washings and comprise sodium perchlorate and ethanol, also more preferably wherein for the second time wash used washings and comprise ethanol), and wash-out (the preferred used elutriant of wash-out comprise the pH damping fluid (as, Tris-HCl)).
Therefore, the present invention preferably provides the method for Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium in the five re-detection samples, and it comprises,
(A) nucleic acid in the extraction sample obtains nucleic acid extraction liquid; With,
(B) nucleic acid extraction liquid is implemented the real-time PCR method of target nucleic acid in the five re-detection samples in single PCR reaction vessel of a first aspect of the present invention.
In this article, " sample " that detects is the potential vitro samples that may contain target nucleic acid or microorganism, such as food, blood, blood products, urine, saliva, medical treatment product or medicine etc.Method of the present invention preferably is not diagnostic method, as the blood sample that can be used for field of public health detect (as, blood sample detects before the blood transfusion, its direct purpose is to judge whether to use this blood sample, but not the patient is diagnosed), and the detection of the product safety of inspection and quarantine field (as, whether clothing pollutes mentioned microorganism, and its direct purpose is the testing product quality).Method of the present invention preferably only limits to the detection to vitro samples, the direct result of detection be target nucleic acid or microorganism existence whether.Even for utilize detection method of the present invention detect pathogenic agent in human or animal's the blood sample (as, virus) target nucleic acid on, whether also can only directly draw the existence of target nucleic acid, also need experienced doctor or sampling personnel to judge that detected target nucleic acid comes from the pathogenic agent that pathogenic agent in the blood or when sampling pollute accidentally, and can not directly obtain diagnostic result or the healthy state of disease; Even target nucleic acid comes from the pathogenic agent in the blood, can only illustrate that also corresponding human or animal is corresponding carrier of pathogens, also need experienced doctor just can judge whether can cause disease or influential to healthy state according to comprehensive conditions such as corresponding human or animal's physique, medical history, clinical symptom.
In this article, " single PCR reaction vessel " refers to that described real-time PCR method carries out technically, there is no need to change container in same container.Most preferably wherein said container is the PCR reaction tubes, and other can carry out the PCR reaction also can carry out the container of real-time fluorescence detection also in protection scope of the present invention.In the real-time PCR method of first aspect present invention, the step that in same container, just can finish pcr amplification and detect in real time, whole process needn't be changed container, thereby has made things convenient for the operator.The operator only need to get final product to adding sample in the container and all ingredients, just can automatically finish by commercially available common real-time fluorescence PCR instrument, and whole process operator only need add sample and reagent gets final product, and is very convenient.Especially, whole process only need be added the step intervention of sample and reagent, be convenient to realize unattended operation, as use the disclosed automatic fine liquid feeding of Chinese patent application 2007100030261 system (namely, the liquid-transfering device of absorption and distribution experimental liquid reaches the PCR instrument with this device), can finish the full process automatization of detection method of the present invention, thereby saved human cost and at utmost reduced the possibility of human error, therefore detection method of the present invention is convenient to cost and reliability advantage that Institute of Automation brings and can be expected.
In this article, " five weights " detects the nucleic acid (or microorganism of its representative) that refers to simultaneously detection five kinds, i.e. Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium.In the real-time fluorescence PCR reaction, the cycle number that experiences during the threshold value of the interior fluorescent signal arrival of each PCR reaction tubes of Ct value representation real-time fluorescence PCR instrument institute default setting, it has good reproducibility, therefore can be used for the good index as sentence read result.In the method for a first aspect of the present invention, for step (3), the Ct value that calculates of a kind of fluoroscopic examination result of target nucleic acid≤45 wherein, then this target nucleic acid is present in the sample; The Ct value that a kind of fluoroscopic examination result of target nucleic acid calculates〉45, then this target nucleic acid is not present in the sample.This is the experience that the long-term lot of experiments of our processes is groped out, and reproducibility and reliability is all good.Therefore also preferred in the method for a first aspect of the present invention, preferably do not adopt internal reference nucleic acid.
The inventor finds, if improve the concentration of probe, can further improve the detection sensitivity of the method for a first aspect of the present invention.So preferably in the method for a first aspect of the present invention, every kind of target nucleic acid probes greater than 5pmol/ml, is preferably 6 ~ 12pmol/ml in the concentration in described single PCR reaction vessel, more preferably 7 ~ 10pmol/ml most preferably is 8pmol/ml.In addition, preferably in the method for a first aspect of the present invention, in single PCR reaction vessel described in the step (1), also further be mixed with other required reagent of PCR reaction, such as salt.Also preferred glycerol concentration is in order to prolong under the PCR condition enzyme resisting power of living.In the specific embodiment of the present invention, salt is Mg salt preferably.Those skilled in the art can also easily select other pH buffer reagents (such as phosphoric acid buffer etc.) and be adjusted to suitable pH, also can easily select other soluble salts (such as KCl etc.) and regulate ionic strength.In addition, can also further be mixed with antioxidant (reductive agent), albumen (enzyme) protective material (as, bovine serum albumin (BSA), human serum albumin (HSA) etc.).The selection of these compositions is all known to those skilled in the art.
In this article, the method for expressing that nucleic acid is commonly used according to this area represents, its sequence is 5 ' end to 3 ' extreme direction as not particularly pointing out.Wherein, be marked with fluorescent marker on the probe.Fluorescent marker can be positioned at 5 ' end of probe, inner and/or 3 ' end, is preferably placed at 5 ' end and/or 3 ' end.In the method for a first aspect of the present invention, probe not of the same race (between the fluoroscopic examination wavelength of fluorophor of institute's mark different, like this can be simultaneously or the different detection wavelength of sequential scanning rapidly, record respectively the change in fluorescence of various fluorophors, thereby can detect simultaneously.In this article, probe has implication well-known to those skilled in the art, and it is by the single stranded DNA for being combined with the target nucleic acid strand that amplifies.Usually, at 5 ' end mark fluorescent group of the nucleotide sequence of every kind of probe, 3 ' end mark quenching group.Preferred wherein various probe marks have different fluorophors.Preferred fluorophor and quenching group thereof are commercially available, as adopting FAM
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Green I,
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ROX
TM/ Texas
With
Deng conventional products, their detection wavelength is different, also can fluorescently-labeled probe be arranged authorized company's complex sign.In the specific embodiment of the present invention, the different fluorescently-labeled probes that detect wavelength that are marked with that wherein adopt all are to entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic, and it is pure that purity reaches HPLC, do not contain assorted band.Preferably in the method for a first aspect of the present invention, various probe marks have different fluorophors.In the specific embodiment of the present invention, preferred fluorophor is FAM, VIC, NED, Texas Red and CY5.
Those skilled in the art can design the PCR reaction conditions according to primer and in requisition for the nucleic acid of pcr amplification.For five re-detections of the present invention, for the balance amplification condition of nucleic acid of the same race not, the inventor is through long-felt, and the condition of optimization is: the condition of each circulation of PCR reaction is 94 ℃ 10 seconds, 55 ℃ 15 seconds and 65 ℃ 45 seconds.In the specific embodiment of the present invention, then preferred first 25 ℃ of insulations of PCR reaction 5 minutes and 94 ℃ of sex change 10 minutes carry out 45 circulations of above-mentioned condition.
In second aspect, the invention provides the detection kit for the real-time PCR method of target nucleic acid in single PCR reaction vessel five re-detection nucleic acid extraction liquid, it comprises archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises
NG-F:ACCGTAACGTCTCTAAGTC，
NG-R:GCAAGATTTCCGATTTGGC，
CT-F:TGTCCATATCTTTGATACGAT，
CT-R:CTCTGATATTTGAAGACTCTACA，
UU-F:CTGCTCGTGAAGTATTACA，
UU-R:GTACCATCTGGGAAAGTAC，
MH-F:TGTGGTTTAATTTGAAGATACAT，
MH-R:AGTCCTCAACTTAATGTTAGA，
MG-F:AGGCAGTAATGTCAATCACA, and
MG-R:GGCTTTATCATTGGCAAACG，
Target nucleic acid probes comprises:
NG-P:CAGACATCACGCACCGAAGCAT，
CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA，
UU-P:ACCAACCATTGTATCTACACCTTCCATA，
MH-P:CCACTCTTGACATCCTTCGCATA, and
MG-P:CCATTCGCATCCAACTTCACCTCT。
In test kit, different reagent can be divided in the different vessels, also can select several prolonged preservation and reagent that chemical reaction do not occur merges and to be kept in the identical container.Container can be the container that bottle, box, syringe etc. can hold mentioned reagent, for example the conventional container that is used for dress PCR, enzyme or nucleic acid reagent.In the detection kit label or specification sheets can also be arranged, operate according to the described method of first aspect present invention in order to indication.Label can be attached on the said vesse, perhaps directly prints on the said vesse, also can provide independently specification sheets.As required, such as the needs that conveniently transport, deposit, test kit further packing advances in the larger packing, and such product also within the scope of the invention.
For the detection kit of second aspect present invention, wherein preferred each composition is preferred as first aspect present invention is described.Preferably such as, wherein various probe marks have different fluorophors, more preferably fluorophor is FAM, VIC, NED, Texas Red and CY5; Wherein every kind of target nucleic acid probes greater than 5pmol/ml, is preferably 6 ~ 12pmol/ml in the concentration in described single PCR reaction vessel, and more preferably 7 ~ 10pmol/ml most preferably is 8pmol/ml.Preferably wherein do not contain internal reference nucleic acid yet.
In the third aspect, the invention provides the application in the detection reagent product of the described test kit of second aspect present invention real-time PCR method of target nucleic acid in for the preparation of five re-detection nucleic acid extraction liquid in single PCR reaction vessel, preferably provide the described detection kit of second aspect present invention for the preparation of the application in the detection reagent product of the described method of first aspect present invention.In this article, the detection reagent product can be detection kit itself, also can be to merge the more bulk goods that a plurality of detection kit are housed.According to preamble, those skilled in the art are readily appreciated that the composition of detection kit wherein and the flow process of method wherein.
Can also comprise the nucleic acid that extracts in the sample in the test kit and obtain the used reagent of nucleic acid extraction liquid.Therefore, in fourth aspect, the invention provides the detection kit for five re-detection sample Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium, it comprises test kit and the required reagent of magnetic bead extraction method of a second aspect of the present invention.
Preferably wherein the required reagent of magnetic bead extraction method comprises,
Nucleic acid extraction liquid, preferred nucleic acid extraction liquid comprise guanidinium isothiocyanate, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl);
For the first time wash used washings, preferably it comprises sodium perchlorate and ethanol;
For the second time wash used washings, preferably it comprises ethanol; With,
The elutriant that wash-out is used, preferably its comprise the pH damping fluid (as, Tris-HCl).
Correspondingly, aspect the 5th, the invention provides the application in the detection reagent of the described test kit of fourth aspect present invention Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium in for the preparation of five re-detection samples.
Aspect the 6th, the invention provides the nucleic acid mixture that does not interfere with each other PCR in real time, it is the mixture of primer or probe, described primer or probe are selected from
NG-F:ACCGTAACGTCTCTAAGTC，
NG-R:GCAAGATTTCCGATTTGGC，
CT-F:TGTCCATATCTTTGATACGAT，
CT-R:CTCTGATATTTGAAGACTCTACA，
UU-F:CTGCTCGTGAAGTATTACA，
UU-R:GTACCATCTGGGAAAGTAC，
MH-F:TGTGGTTTAATTTGAAGATACAT，
MH-R:AGTCCTCAACTTAATGTTAGA，
MG-F:AGGCAGTAATGTCAATCACA，
MG-R:GGCTTTATCATTGGCAAACG，
NG-P:CAGACATCACGCACCGAAGCAT，
CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA，
UU-P:ACCAACCATTGTATCTACACCTTCCATA，
MH-P:CCACTCTTGACATCCTTCGCATA, and
MG-P:CCATTCGCATCCAACTTCACCTCT。
This mixture can be used for the method for a first aspect of the present invention, also can be for the preparation of the test kit of the present invention second and/or fourth aspect.
Beneficial effect of the present invention is: can whether have Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium in the five re-detection samples, five heavy primer/probes are without crossed contamination and interference, the accuracy, reliability, specificity, sensitivity and the repeatability that have guaranteed, and the present conventional commercially available real-time fluorescence PCR equipment of use, need not to transform, easy to operate and save cost, and can not use the internal reference monitoring, further saved cost.
For the ease of understanding, below will describe in detail the present invention by concrete accompanying drawing, embodiment.It needs to be noted, these descriptions only are exemplary descriptions, do not consist of limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change all are apparent concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is excessively the same in this article for their full text.
Description of drawings
Fig. 1 has shown the detection collection of illustrative plates that in the exemplary embodiment of the invention Diplococcus gonorrhoeae (NG), chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), mycoplasma hominis (MH), mycoplasma genitalium (MG) is carried out five heavy PCR in real time, and its result curve can clearly be distinguished.
Embodiment
Following this paper will describe invention by specific embodiment.As do not specialize part, can be according to " molecule can swell experiment guide " (third edition) (Cold Spring Harbor laboratory Press), " cell experiment guide " (Science Press that those skilled in the art were familiar with, Beijing, China, calendar year 2001), " RNA experimental technique handbook (Science Press, Beijing, China, 2004), " immunoassay technology " (Science Press, Beijing, China, 1991) etc. listed method is implemented in the reference quoted of laboratory manual and this paper.Wherein, used probe, primer can entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic.
The extraction of embodiment 1 nucleic acid
Diplococcus gonorrhoeae (NG), chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), mycoplasma hominis (MH) and mycoplasma genitalium (MG) can be standard substance available from health ministry visiting center under permissive condition, for nucleic acid extraction.
The extraction of nucleic acid is extracted according to conventional magnetic bead extraction method, nucleic acid extraction for the above-mentioned five kinds of microorganisms of simultaneous adaptation, improve as follows: add 90uL nucleic acid extraction liquid (prescription and final concentration are: guanidinium isothiocyanate 1.2M, sodium ethylene diamine tetracetate (pH8.0) 10mM, tween 20 2%(W/W), sodium perchlorate 1M, ethanol 40%(V/V to the 1uL standard substance), Tris-HCl(pH8.0) 10mM), in 42 ℃ of insulation 10min, then add 10uL
D-Beads DNA bead suspension (50mg/mL, can be available from Beijing beguine Bioisystech Co., Ltd that ends), after vibration mixes, be enclosed within and apply magnetic field on the magnet stand, discard wherein liquid, then (prescription and final concentration are: sodium perchlorate 1M to add 200uL washings A, ethanol 30%(V/V)), discard washings A after the washing, adding 200uL washings B(prescription and final concentration is again: ethanol 70%(V/V)), discard washings B after the washing, (prescription and final concentration are: Tris-HCl(pH8.0) 10mM), be incubated 10min in 42 ℃ to add at last elutriant, sucking-off and retaining liquid are nucleic acid extraction liquid.
The nucleic acid extraction liquid that merges each microbial standard product, and dilution is for following step test.
Embodiment 2 five heavy PCR in real time tests
1, primer and probe sequence
Entrust synthetic following primer pair and probe:
Detect the primer pair of Diplococcus gonorrhoeae:
NG-F:ACCGTAACGTCTCTAAGTC
NG-R:GCAAGATTTCCGATTTGGC
Detect the probe of Diplococcus gonorrhoeae:
NG-P:CAGACATCACGCACCGAAGCAT
Detect the primer pair of chlamydia trachomatis:
CT-F:TGTCCATATCTTTGATACGAT
CT-R:CTCTGATATTTGAAGACTCTACA
Detect the probe of chlamydia trachomatis:
CT-P:CCAAGCCGAGTCTACAGTTATAGGTCA
Detect the primer pair of Ureaplasma urealyticum:
UU-F:CTGCTCGTGAAGTATTACA
UU-R:GTACCATCTGGGAAAGTAC
Detect the probe of Ureaplasma urealyticum:
UU-P:ACCAACCATTGTATCTACACCTTCCATA
Detect the primer pair of mycoplasma hominis:
MH-F:TGTGGTTTAATTTGAAGATACAT
MH-R:AGTCCTCAACTTAATGTTAGA
Detect the probe of mycoplasma hominis:
MH-P:CCACTCTTGACATCCTTCGCATA
Detect the primer pair of mycoplasma genitalium:
MG-F:AGGCAGTAATGTCAATCACA
MG-R:GGCTTTATCATTGGCAAACG
Detect the probe of mycoplasma genitalium:
MG-P:CCATTCGCATCCAACTTCACCTCT
2, fluorescent mark
5 ' end at above-mentioned probe CT-P, NG-P, UU-P, MH-P and MG-P is entrusted respectively complex sign fluorescent mark FAM, VIC, NED, Texas Red and CY5, and at the corresponding quenching group of 3 ' end mark.
3, PCR reaction conditions
PCR reaction system cumulative volume is 20 μ l, wherein the final concentration of each component is respectively: nucleic acid extraction liquid (100 times of dilutions) 1uL, each primer content in above-mentioned 5 pairs of primer pairs is 15pmol/ml, and each content in above-mentioned 5 kinds of probes increases to 8pmol/ml, Mg
2+(MgCl
2) concentration is 3.75mmol/ml, dNTP concentration respectively is 0.2mmol/ml, and UNG enzyme content is 0.05U, and 2 * PCR buffer(can be available from TaKaRa company, and pH8.3 is without Mg
2+) be 10ul, the Taq archaeal dna polymerase is 2U, glycerine 15%(V/V), surplus is deionized water.
The reaction heat cycling condition is as follows:
25 ℃ 5 minutes 94 ℃ 10 minutes
(94 ℃ 10 seconds 55 ℃ 15 seconds 65 ℃ 45 seconds) * 45 circulations
Use ABI 7500 real-time fluorescence PCR instrument, fluorescent collecting FAM, VIC, NED, Texas Red and CY5 detect wavelength.
4, the result judges
Baseline and threshold setting are ABI 7500 luminoscopes acquiescence automatic setting.If each fluorescence (FAM, VIC, NED, Texas Red or CY5) layer Ct value is greater than 45, the detection of nucleic acids that then is judged to be corresponding microorganism is negative, if less than or equal to 45, judges that then the detection of nucleic acids of corresponding microorganism is positive.The concrete collection of illustrative plates that detects is seen Figure 15, shows that method of the present invention can detect the target nucleic acid of the mentioned microorganism of concentration at the bottom of the utmost point, and detects and do not disturb mutually, has reached super quick detection sensitivity.
Above test repeats 120 times and (comprises and only add one, two, three or the nucleic acid extraction liquid of four kind of microbial standard product in the sample, perhaps do not add the situation of any extracting solution, but other testing conditions are constant), the result is all correct, show accuracy of the present invention and good reliability, need not internal reference.
Sequence table
＜110〉Suzhou Hua Yimei bio tech ltd
＜120〉five fast super quick detection kit and application thereof of heavy fluorescent PCR
＜130〉China's application
<160>15
<170>PatentIn version 3.5
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accgtaacgt ctctaagtc 19
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gcaagatttc cgatttggc 19
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tgtccatatc tttgatacga t 21
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ctctgatatt tgaagactct aca 23
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ctgctcgtga agtattaca 19
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gtaccatctg ggaaagtac 19
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tgtggtttaa tttgaagata cat 23
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agtcctcaac ttaatgttag a 21
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aggcagtaat gtcaatcaca 20
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ggctttatca ttggcaaacg 20
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cagacatcac gcaccgaagc at 22
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ccaagccgag tctacagtta taggtca 27
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accaaccatt gtatctacac cttccata 28
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ccactcttga catccttcgc ata 23
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ccattcgcat ccaacttcac ctct 24
Claims (16)
1. the real-time PCR method of target nucleic acid in the five re-detection nucleic acid extraction liquid in single PCR reaction vessel, described target nucleic acid be from Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium, and it comprises,
(1) mixed nucleus acid extraction liquid, archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes in described single PCR reaction vessel, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises
ACCGTAACGTCTCTAAGTC，
GCAAGATTTCCGATTTGGC，
TGTCCATATCTTTGATACGAT，
CTCTGATATTTGAAGACTCTACA，
CTGCTCGTGAAGTATTACA，
GTACCATCTGGGAAAGTAC，
TGTGGTTTAATTTGAAGATACAT，
AGTCCTCAACTTAATGTTAGA，
AGGCAGTAATGTCAATCACA, and
GGCTTTATCATTGGCAAACG，
Target nucleic acid probes comprises:
CAGACATCACGCACCGAAGCAT，
CCAAGCCGAGTCTACAGTTATAGGTCA，
ACCAACCATTGTATCTACACCTTCCATA，
CCACTCTTGACATCCTTCGCATA, and
CCATTCGCATCCAACTTCACCTCT；
(2) carry out the PCR reaction, and detect in real time the fluorescence of different wave length; With,
(3) the Ct value that calculates according to the fluoroscopic examination result has judged whether that target nucleic acid is present in the sample.
2. the method for Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium in the five re-detection samples, it comprises,
(A) nucleic acid in the extraction sample obtains nucleic acid extraction liquid; With,
(B) to nucleic acid extraction liquid implement claimed in claim 1 in single PCR reaction vessel the real-time PCR method of target nucleic acid in the five re-detection samples.
3. claim 1 or 2 described methods, it is non-diagnostic method.
4. method claimed in claim 1, wherein various probe marks have different fluorophors, and preferred fluorophor is FAM, VIC, NED, Texas Red and CY5.
5. method claimed in claim 1, wherein the condition of each circulation of PCR reaction is 94 ℃ 10 seconds, 55 ℃ 15 seconds and 65 ℃ 45 seconds.
6. method claimed in claim 1, wherein every kind of target nucleic acid probes greater than 5pmol/ml, is preferably 6 ~ 12pmol/ml in the concentration in described single PCR reaction vessel, and more preferably 7 ~ 10pmol/ml most preferably is 8pmol/ml.
7. method claimed in claim 2, the nucleic acid that wherein extracts in the sample is to adopt the magnetic bead extraction method to extract.
8. method claimed in claim 7, the process of the nucleic acid in the test product of wherein getting sample is: (the preferred nucleic acid extraction liquid comprises guanidinium isothiocyanate to add the nucleic acid extraction liquid to sample, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl)), add magnetic bead after the insulation, after mixing, after applying magnetic field, discard wherein liquid, then washing (preferred washed twice, more preferably wherein for the first time wash used washings and comprise sodium perchlorate and ethanol, also more preferably wherein for the second time wash used washings and comprise ethanol), and wash-out (the preferred used elutriant of wash-out comprise the pH damping fluid (as, Tris-HCl)).
9. the detection kit that is used for the real-time PCR method of target nucleic acid in single PCR reaction vessel five re-detection nucleic acid extraction liquid, it comprises archaeal dna polymerase, dNTP, target nucleic acid primer pair and target nucleic acid probes, wherein said probe mark has fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different, wherein
The target nucleic acid primer pair comprises
ACCGTAACGTCTCTAAGTC，
GCAAGATTTCCGATTTGGC，
TGTCCATATCTTTGATACGAT，
CTCTGATATTTGAAGACTCTACA，
CTGCTCGTGAAGTATTACA，
GTACCATCTGGGAAAGTAC，
TGTGGTTTAATTTGAAGATACAT，
AGTCCTCAACTTAATGTTAGA，
AGGCAGTAATGTCAATCACA, and
GGCTTTATCATTGGCAAACG，
Target nucleic acid probes comprises:
CAGACATCACGCACCGAAGCAT，
CCAAGCCGAGTCTACAGTTATAGGTCA，
ACCAACCATTGTATCTACACCTTCCATA，
CCACTCTTGACATCCTTCGCATA, and
CCATTCGCATCCAACTTCACCTCT。
10. test kit claimed in claim 9, wherein various probe marks have different fluorophors, and preferred fluorophor is FAM, VIC, NED, Texas Red and CY5.
11. test kit claimed in claim 9, wherein every kind of target nucleic acid probes greater than 5pmol/ml, is preferably 6 ~ 12pmol/ml in the concentration in described single PCR reaction vessel, and more preferably 7 ~ 10pmol/ml most preferably is 8pmol/ml.
12. be used for the detection kit of five re-detection sample Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium, it comprises arbitrary described test kit and the required reagent of magnetic bead extraction method of claim 9 ~ 11.
13. the described test kit of claim 12, wherein the required reagent of magnetic bead extraction method comprises,
Nucleic acid extraction liquid, preferred nucleic acid extraction liquid comprise guanidinium isothiocyanate, sodium ethylene diamine tetracetate, tween 20, sodium perchlorate, ethanol and pH damping fluid (as, Tris-HCl);
For the first time wash used washings, preferably it comprises sodium perchlorate and ethanol;
For the second time wash used washings, preferably it comprises ethanol; With,
The elutriant that wash-out is used, preferably its comprise the pH damping fluid (as, Tris-HCl).
14. the application of the detection reagent product of the arbitrary described test kit of claim 9 ~ 11 real-time PCR method of target nucleic acid in for the preparation of five re-detection nucleic acid extraction liquid in single PCR reaction vessel (the detection reagent product that preferably is used for arbitrary described method of claim 1-8).
15. the application of the detection reagent of claim 12 or 13 described test kits Diplococcus gonorrhoeae, chlamydia trachomatis, Ureaplasma urealyticum, mycoplasma hominis and mycoplasma genitalium in for the preparation of five re-detection samples (the detection reagent product that preferably is used for claim 2,7 or 8 described methods).
16. do not interfere with each other the nucleic acid mixture of PCR in real time, it is the mixture of primer or probe, described primer or probe are selected from
ACCGTAACGTCTCTAAGTC，
GCAAGATTTCCGATTTGGC，
TGTCCATATCTTTGATACGAT，
CTCTGATATTTGAAGACTCTACA，
CTGCTCGTGAAGTATTACA，
GTACCATCTGGGAAAGTAC，
TGTGGTTTAATTTGAAGATACAT，
AGTCCTCAACTTAATGTTAGA，
AGGCAGTAATGTCAATCACA，
GGCTTTATCATTGGCAAACG，
CAGACATCACGCACCGAAGCAT，
CCAAGCCGAGTCTACAGTTATAGGTCA，
ACCAACCATTGTATCTACACCTTCCATA，
CCACTCTTGACATCCTTCGCATA, and
CCATTCGCATCCAACTTCACCTCT。
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| CN111321206A (en) * | 2020-03-06 | 2020-06-23 | 杭州博日科技有限公司 | Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof |
| CN111321206B (en) * | 2020-03-06 | 2023-09-22 | 杭州博日科技股份有限公司 | Detection system of quintuple fluorescent PCR (polymerase chain reaction), application and product thereof |
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