CN101624629B - PCR detection method of multiple-target nucleic acid in single pipe and kit thereof - Google Patents
PCR detection method of multiple-target nucleic acid in single pipe and kit thereof Download PDFInfo
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Abstract
The invention relates to a real-time PCR method of one or more kinds of target nucleic acid in multiple-detection samples in a signal PCR reaction vessel, which comprises the following steps: (1) mixing a sample, DNA polymerase, dNTP, internal contrast nucleic acid, a target nucleic acid primer pair, an internal contrast nucleic acid primer pair, one or more kinds of target nucleic acid probes and internal contrast nucleic acid probes in the signal PCR reaction vessel, wherein the probes are marked with fluorescent groups and quenching groups, and moreover, the fluoroscopic detection wavelengths of the fluorescent groups marked by various probes are different; (2)detecting fluorescent light with different wave lengths in real time to carry out the PCR reaction; (3) calculating a Ct value according to a fluorescent light detecting result to judge whether one or more kinds of target nucleic acid is stored in the sample; in additon, the invention also discloses a detection kit for the method and preparation, application, and the like of the corresponding detection kit. The multiple-detection method does not need special devices and can be widely used in the fields of laboratory investigation, food security, medicine, hygiene, and the like.
Description
Technical field
The invention belongs to the nucleic acid detection technique field, particularly, the present invention relates in single PCR reaction vessel the PCR method of target nucleic acid in the multiple test sample.In addition, the invention still further relates to reagent related in the above-mentioned PCR method,, and be used for the detection kit of aforesaid method and the preparation application of corresponding detection kit etc. like internal reference, primer, probe etc.
Background technology
Polymerase chain reaction (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., and the function at aspects such as detection of nucleic acids, clinical diagnosis and molecular biology researches seems more next important.The present Application Areas of this type technology is very extensive, in numerous industries such as laboratory study, food safety, medical and health, is able to use.Except the PCR in real time detection of routine, people detect PCR in real time and have also done a large amount of improvements in recent years.For example, one Chinese patent application CN1768151A, CN101189505A, CN101273262A, CN101302473A etc. have all put down in writing the improvement to traditional PCR in real time.But traditional and improved PCR in real time (especially real-time fluorescence PCR) even only detect single target nucleic acid, also must be faced the interference of the multiple nucleotide pair detected result that possibly exist in the sample when carrying out the detection of actual sample; When in single PCR reaction vessel, carrying out multiple-target nucleic acid detection (promptly detecting multiple target nucleic acid simultaneously); Owing to will introduce the detection not primer and the probe of target nucleic acid of the same race simultaneously, therefore also further demand side is to the mutual interferential problem of different primers and probe.And in the face of these difficult problems; Often adopt complicated operation, processing means that cost is high in the prior art; Even some can't introduce the internal reference Monitoring systems and get rid of false negative, the internal reference of perhaps introducing (molecule) itself (normally nucleic acid) further aggravated with multiple target nucleic acid, primer and probe thereof between mutual interference mutually.
In order to overcome the deficiency of prior art; Through long-term and arduous effort; The inventor is surprisingly through preferred result method of discrimination, optimization sample treatment, selection internal reference gene and the optional fluorescently-labeled probe that is packaged into virus-like particle and/or has filtered out primer and different wavelength regions; Develop the PCR method of target nucleic acid in multiple test sample in the single PCR reaction vessel (like, single PCR reaction tubes), overcome the easy crossed contamination of multi-primers/probe and the low relatively technological difficulties of sensitivity that generally run in the real-time fluorescence PCR exploitation; And detect for single tube; Easy to operate and save cost, adopt omnidistance internal reference supervisory system simultaneously, the accuracy of warranty test.Through the checking of great amount of samples, reliable results, and use conventional at present commercially available real-time fluorescence PCR equipment, and need not to transform, technical indicators such as its specificity, sensitivity, repeatability are all reached advanced world standards.In addition, the inventor has also invented the reagent that is used for aforesaid method, like internal reference, primer, probe etc., and the preparation application of detection kit and corresponding detection kit etc.
Summary of the invention
The object of the present invention is to provide in single PCR reaction vessel the real-time PCR method of target nucleic acid in the multiple test sample; This method through the optimum result method of discrimination, optimize sample treatment, optimize the internal reference gene and optimize its packaged form and/or optimize and non-interfering primer, probe and fluorescent mark; Can in single PCR reaction vessel, tell the target nucleic acid not of the same race that whether exists in the sample simultaneously; Especially can tell RNA and DNA target nucleic acid simultaneously; And need not Special Equipment or reagent, and also need not to introduce complicated experimental procedure, just can obtain the result of high specific, sensitivity and repeatability.In addition, the present invention also aims to provide the detection kit of aforesaid method and the preparation application of corresponding detection kit etc.
Particularly, in first aspect, the invention provides in single PCR reaction vessel the real-time PCR method of one or more target nucleic acids in the multiple test sample, it comprises,
(1) in said single PCR reaction vessel biased sample, archaeal dna polymerase, dNTP (being dATP, dCTP, dGTP and dTTP), internal reference nucleic acid, target nucleic acid primer to, internal reference nucleic acid primer to, one or more target nucleic acid probes and internal reference nucleic probe; Wherein aforementioned various probe marks have fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different;
(2) carry out the PCR reaction, and detect the fluorescence of different wave length in real time; With
(3) the Ct value that calculates according to the fluoroscopic examination result has judged whether that one or more target nucleic acids are present in the sample.
In first aspect of the present invention, the sample that said real-time PCR method detected is the potential vitro samples that possibly contain target nucleic acid, like food, blood, blood products, saliva, medical treatment product or medicine etc.Said real-time PCR method only limits to the detection to vitro samples, the direct result of detection be target nucleic acid 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; The existence that also can only directly draw target nucleic acid whether; Also need empirical doctor or sampling personnel and judge that detected target nucleic acid comes from the pathogenic agent that pathogenic agent or when sampling in the blood pollute accidentally, and can not directly obtain the diagnostic result or the healthy state of disease; Even target nucleic acid comes from the pathogenic agent in the blood; Can only explain that also corresponding human or animal is corresponding carrier of pathogens, also need empirical doctor and just can judge whether can cause disease or influential healthy state according to comprehensive conditions such as corresponding human or animal's physique, medical history, clinical symptom.Preferably in the method for first aspect of the present invention, sample be through pre-treatment and enrichment the sample of nucleic acid (like target nucleic acid), be the sample of handling through the magnetic bead extraction method like sample.Nonspecific magnetic bead extraction method adopts the surface to scribble the paramagnetic particle of non-specific nucleic acid absorption type material; Low pH (as; PH value 5～7) nucleic acid can be adsorbed onto the paramagnetic particle surface and under the situation of high salt concentration, carry out that magnetic separates and thorough washing after, high pH (as; PH value 8～9), can nucleic acid be eluted under the condition of low salt concn, thus enrichment the sample of nucleic acid (like target nucleic acid) can be used for the PCR test; Can also adopt the magnetic bead extraction method of specific adsorption (like hybridization absorption and immunosorption) in addition.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. Chinese law medical journal, 18 (3): 107-108; One Chinese patent application 200610030229.5 etc.
In this article, " single PCR reaction vessel " refers to that said 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 PCR reaction and can carry out container that real-time fluorescence detects also in protection scope of the present invention.In the real-time PCR method of first aspect present invention, in same container, just can accomplish pcr amplification and the step that detects in real time, whole process needn't be changed container, thereby has made things convenient for the operator.The operator only need in container, add sample and all ingredients gets final product, and just can come to accomplish automatically through commercially available common real-time fluorescence PCR appearance, 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 one Chinese patent application 2007100030261 disclosed automatic trace to add liquid system (that is, draw and distribute the liquid-transfering device of experimental liquid and have the PCR appearance of this device), can accomplish the full process automatization of detection method of the present invention; Thereby practiced thrift human cost and at utmost reduced the possibility of human error, therefore detection method of the present invention is convenient to cost and safety advantage that Institute of Automation brings and can be expected.
In this article, " multiple detection " refer to simultaneously the nucleic acid that detects have multiple, promptly at least two kinds.In the method for first aspect of the present invention, owing to detected internal reference nucleic acid and a kind of target nucleic acid at least, the nucleic acid that therefore detects has two kinds at least.When the target nucleic acid kind that will detect more than when a kind of, and/or more than when a kind of as the internal reference nucleic acid species of internal reference, the corresponding increase of nucleic acid that then detects simultaneously.Wherein, every kind of nucleic acid can be RNA, also can be DNA.In embodiment of the present invention, preferably carry out two re-detections, three re-detections, quadruple detection or five re-detections.Preferred existing RNA target nucleic acid also has the DNA target nucleic acid in the multiple target nucleic acid.Equally, preferred existing RNA internal reference nucleic acid also has DNA internal reference nucleic acid in the multiple internal reference nucleic acid.
Preferably in the method for 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, like pH buffer reagent and salt.In addition, also preferred glycerol concentration is in order to prolong under the PCR condition enzyme resisting power of living.In embodiment of the present invention, the pH buffer reagent is Tris.HCl preferably; Salt is KCl preferably.Those skilled in the art can also easily select other pH buffer reagents (like phosphoric acid buffer etc.) and be adjusted to suitable pH, also can easily select other soluble salts (like magnesium chloride etc.) and regulate ionic strength.In addition, can also further be mixed with inhibitor (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.
Preferably in the method for first aspect of the present invention; In single PCR reaction vessel described in the step (1), also further be mixed with ThermoScript II; For example, common ThermoScript II has avian myeloblastosis virus (AMV) ThermoScript II or Moloney murine leukemia virus (MLV) ThermoScript II.Like this, the method for first aspect of the present invention in order to DNA amplification class target nucleic acid and internal reference nucleic acid, and can be carried out reverse transcription PCR (RT-PCR), in order to cloning RNA class target nucleic acid and internal reference nucleic acid except carrying out conventional PCR reaction (amplification).At this moment, the PCR reaction carried out of step (2) is exactly the RT-PCR reaction.More preferably also being mixed with the RNA enzyme inhibitors in the single PCR reaction vessel described in the step (1), like RNasin.Like this, in the RT-PCR reaction, can prevent the degraded of RNA template.
Preferably in the method for first aspect of the present invention; Wherein target nucleic acid preferably have a kind of, two or three, wherein every kind of target nucleic acid is respectively RNA or DNA, preferably pathogenic agent RNA or DNA; Be more preferably viral RNA or DNA; As preferably HCV (hepatitis C virus, it is a RNA viruses) RNA, HIV (human immunodeficiency virus, it is a RNA viruses) RNA or HBV (hepatitis B virus; It is a dna virus) DNA most preferably is 5 ' the non-coding region RNA of HCV, the gag gene RNA of HIV or the antigenic gene DNA of coding S of HBV.For example, the target nucleic acid of detection can be among HCV RNA, HIV RNA and the HBV DNA a kind of, two or three.In embodiment of the present invention, the example of target nucleic acid has 5 ' the non-coding region RNA of HCV, the gag gene RNA of HIV and the antigenic gene DNA of coding S of HBV.
In the method for first aspect of the present invention, internal reference nucleic acid is the nucleic acid of debond target nucleic acid probes, preferably neomycin resistance gene or its fragment, and also preferred internal reference nucleic acid is packaged in the virus-like particle.The kind of the internal reference nucleic acid preferably kind with target nucleic acid is identical.As, target nucleic acid is DNA, then corresponding internal reference nucleic acid is RNA; And target nucleic acid is RNA, and then corresponding internal reference nucleic acid is DNA; Existing DNA also has RNA in the multiple target nucleic acid, and then internal reference nucleic acid has two kinds, is respectively DNA and RNA, is respectively applied for the internal reference as DNA and RNA class target nucleic acid.The inventor puts into practice through the comparison and long-term detection the in nucleic acid sequence data storehouse; The sequence difference of the antigenic gene DNA of coding S of 5 ' the non-coding region RNA of discovery neomycin resistance gene and HCV, the gag gene RNA of HIV and HBV is very big; The other side's probe is difficult to interosculate; Thereby be difficult to interfere with each other, thereby can be used for as the contrast of effectively getting rid of false negative result.The nucleotide sequence of preferred DNA internal reference nucleic acid comprises the sequence shown in the Seq ID NO:16 or shown in Seq ID NO:16; The nucleotide sequence of preferred RNA internal reference nucleic acid comprises the sequence shown in the Seq ID NO:17 or shown in Seq ID NO:17.In order to make internal reference nucleic acid be similar to pathogenic agent more; Preferred internal reference nucleic acid is packaged in the virus-like particle (comprising pseudovirus); Preferably be packaged in the phage virus-like particle; May be packaged among MS2 phage or the TMV etc. like RNA internal reference nucleic acid, DNA internal reference nucleic acid may be packaged in M13 phage etc.Known these packing techniques of those skilled in the art are as can be referring to Zhang Yusong etc., the more recent application progress of pseudovirus.The medical research magazine, 37 (6): 116-118; Zhang Kuo etc., RNA phage virus-like particle packing mechanism and Research development thereof. mikrobe and infection, 3 (2): 111-114; Chen Xinzhong etc., real-time fluorescence quantitative RT-PCR detects the cabrilla viral nervous necrosis. hi-tech communication, 16 (4): 431-434; 18 (3): 107-108 etc.
In the method for first aspect of the present invention; The fluoroscopic examination wavelength of the fluorophor of institute's mark is different between the probe not of the same race (comprising target nucleic acid probes not of the same race, internal reference nucleic probe not of the same race); Like this can be simultaneously or the wavelength of sequential scanning different detection apace; Note the change in fluorescence of various fluorophors respectively, thereby can detect simultaneously.In this article, probe has implication well-known to those skilled in the art, its by for can with the target nucleic acid strand bonded single stranded DNA 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
TM/
Green I,
/ JOE/HEX, NED
TM/ TAMRA
TM/
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 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's synthetic, and it is pure that purity reaches HPLC, do not contain assorted band.
For the right target nucleic acid of preferred pin in the method for first aspect of the present invention; Also preferred pin is to the nucleotide sequence of these preferred target nucleic acid design target nucleic acid probes; More preferably the nucleotide sequence of target nucleic acid probes is selected from continuous sequence or its complementary sequence in the gag gene of continuous sequence or its complementary sequence and HIV in the 5 ' non-coding region of continuous sequence or its complementary sequence in the antigenic gene of coding S of HBV, HCV, most preferably is selected from Seq ID No:11 or its complementary sequence, Seq ID No:12 or its complementary sequence and Seq ID No:13 or its complementary sequence.When detecting at the same time, the nucleotide sequence of these most preferred probes can not combine other target nucleic acids and internal reference nucleic acid.Correspondingly, for the right target nucleic acid of preferred pin in the method for first aspect of the present invention, also preferred pin is right to these preferred target nucleic acid design primers.In this article, primer is to having implication well-known to those skilled in the art, and it is made up of upstream primer that is respectively single stranded DNA and downstream primer, and corresponding gene or its fragment can be used to increase.The antigenic gene of coding S that gene that preferred target nucleic acid primer is increased or fragment are selected from HBV or among, the 5 ' non-coding region of HCV or among and the gag gene of HIV or among, most preferred target nucleic acid primer is to being selected from Seq IDNo:1 and 2, Seq ID No:3 and 4 and Seq ID No:5 and 6.
In addition; For the preferred internal reference nucleic acid that uses in the method for first aspect of the present invention; Also preferred pin is to the nucleotide sequence of these preferred internal reference nucleic acid design internal reference nucleic probes; More preferably the nucleotide sequence of internal reference nucleic probe is selected from continuous sequence or its complementary sequence in the neomycin resistance gene; Most preferably the nucleotide sequence of internal reference nucleic probe is SeqID No:14 or 15, and Seq ID No:14 is to DNA internal reference nucleic acid, and Seq ID No:15 is to RNA internal reference nucleic acid.When detecting at the same time, the nucleotide sequence of these most preferred probes can not combine other internal reference nucleic acid and target nucleic acids.Correspondingly, for the preferred internal reference nucleic acid that uses in the method for first aspect of the present invention, also preferred pin is right to these preferred internal reference nucleic acid design primers.Gene that preferred internal reference nucleic acid primer is increased or fragment be selected from the neomycin resistance gene or among, most preferred internal reference nucleic acid primer is to being selected from SeqIDNo:7 and 8 and SeqIDNo:9 and 10.
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 multiple detection, for the balance amplification condition of nucleic acid of the same race not, the inventor is through long-felt, optimizes the condition that and is: each round-robin condition of PCR reaction is 94 ℃ 10 seconds, 50 ℃ 10 seconds and 65 ℃ 45 seconds.In embodiment of the present invention, 45 circulations of above-mentioned condition are carried out in preferred 50 ℃ of insulations earlier of PCR reaction 2 minutes and 94 ℃ of sex change 3 minutes then; Carry out the reaction of RT-PCR for needs, before aforementioned PCR reaction, also carry out 42 ℃ of insulations 30 minutes, to carry out reverse transcription.
In the real-time fluorescence PCR reaction, the cycle number that is experienced during the threshold value of the interior fluorescent signal arrival of each PCR reaction tubes of Ct value representation real-time fluorescence PCR appearance institute default setting, it has good reproducibility, therefore can be used for the good index as sentence read result.The threshold value of default setting is preferably 10 times of standard deviation of 3～15 round-robin fluorescent signals.Preferably in the method for first aspect of the present invention, for step (3), Ct value≤45 that calculate of a kind of fluoroscopic examination result of target nucleic acid wherein, then this target nucleic acid is present in the sample; Ct value＞45 that a kind of fluoroscopic examination result of target nucleic acid calculates, and Ct value＜30 that calculate of the fluoroscopic examination result of corresponding internal reference nucleic acid, then this target nucleic acid is not present in the sample.This is the experience that we grope out through long-term a large amount of tests.Other situation, i.e. Ct value >=30 that Ct value＞45 that calculate of a kind of fluoroscopic examination result of target nucleic acid, and the fluoroscopic examination result of corresponding internal reference nucleic acid calculate then need remeasure.So simple interpretation can accomplish with manual work, avoided the very complicated reagent or the use of instrument.
In second aspect; The invention provides the detection kit that is used for the said method of first aspect present invention; It comprises archaeal dna polymerase, dNTP, internal reference nucleic acid, target nucleic acid primer to, internal reference nucleic acid primer to, one or more target nucleic acid probes and internal reference nucleic probe; Wherein aforementioned various probe marks have fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different.Wherein, different reagent can be divided in the different vessels, also can select several kinds of prolonged preservation and reagent that chemical reaction do not take place merges and to be kept in the identical container.Container can be the container that bottle, box, syringe etc. can hold mentioned reagent, the for example conventional container that is used to adorn PCR, enzyme or nucleic acid reagent.In the detection kit label or specification sheets can also be arranged, operate according to the said 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 independently specification sheets can be provided.As required, like the needs that conveniently transport, deposit, test kit further packing advances in the bigger 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 also further comprise ThermoScript II; Wherein also further comprise the required reagent of magnetic bead extraction method, like magnetic bead, lysate, washings etc.; Wherein internal reference nucleic acid is neomycin resistance gene or its fragment, and preferred said gene or its fragment are packaged in the virus-like particle; Various probe marks have different fluorophors; Wherein the nucleotide sequence of target nucleic acid probes is selected from continuous sequence or its complementary sequence in the antigenic gene of coding S of continuous sequence or its complementary sequence in the gag gene of continuous sequence or its complementary sequence in the 5 ' non-coding region of HCV, HIV, HBV; And/or wherein the nucleotide sequence of internal reference nucleic probe is selected from continuous sequence or its complementary sequence in the neomycin resistance gene.Most preferably wherein the nucleotide sequence of internal reference nucleic acid, various probe, various primers is selected from the sequence in the appended sequence table of this specification sheets.
In the third aspect, the invention provides the described detection kit of second aspect present invention is used for the detection reagent product of the said method of first aspect present invention in preparation application.The detection reagent product can be a 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.
For the ease of understanding, below will describe in detail the present invention through concrete accompanying drawing, embodiment.What need particularly point out is that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change all are conspicuous 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 the same excessively in this article for their full text.
Description of drawings
Fig. 1 has shown the detection collection of illustrative plates of the double PCR in real time in the exemplary embodiment of the invention.
Fig. 2 has shown the detection collection of illustrative plates of the triple PCR in real time in the exemplary embodiment of the invention.
Fig. 3 has shown the detection collection of illustrative plates of the quadruple PCR in real time in the exemplary embodiment of the invention.
Fig. 4 has shown the detection collection of illustrative plates of the heavy PCR in real time of five in the exemplary embodiment of the invention.
Embodiment
Following this paper will describe invention through concrete 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, Beijing that those skilled in the art were familiar with; China; Calendar year 2001), " RNA experimental technique handbook (Science Press, Beijing, China; 2004), " immunoassay technology " (Science Press; Beijing, China, 1991) etc. in the reference that laboratory manual and this paper quoted listed method implement.Wherein used probe, primer can entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthetic.
1.DNA type dual Real Time of target nucleic acid (in real time) PCR reaction
Be used to detect HBV DNA and DNA internal reference nucleic acid (Seq ID No:16)
Primer
The primer of amplification HBV DNA is HBV-FQ-L1 (Seq ID No:1) and HBV-FQ-R1 (Seq ID No:2); The primer of DNA amplification internal reference nucleic acid is IC309L1 (Seq ID No:7) and IC309R1 (Seq ID No:8)
Fluorescent probe
The nucleotides sequence of the fluorescent probe of HBV is classified HBV-FQ-P1 (Seq ID No:11) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of DNA internal reference nucleic probe is classified IC-FQ-P1 (Seq IDNo:14) as, and its 5 ' end adopts the ROX mark, the corresponding quenching group of 3 ' end mark.
The PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acid samples are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 50 |
94 ℃ 3 minutes | |||
| (94 |
50 |
65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence gathers FAM and ROX detects wavelength.
The result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and ROX layer Ct value＜30, then be judged to be HBV DNA feminine gender; Like FAM layer Ct value＞45 and ROX layer Ct value >=30, then need heavily inspection;
Like FAM layer Ct value≤45, then be judged to be the HBV DNA positive.The concrete collection of illustrative plates that detects is shown in Figure 1A.
2.RNA type dual real-time PCR reactions of target nucleic acid
Be used to detect HCV RNA and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq ID No:17)
Primer
The primer of amplification HCV RNA is HCV-F-L1 (Seq ID No:3) and HCV-F-R1 (Seq ID No:4); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
Fluorescent probe
The nucleotides sequence of the fluorescent probe of HCV is classified HCV-FQ-P1 (Seq ID No:12) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
The PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acid samples are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 |
50 |
65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence gathers FAM and HEX detects wavelength.
The result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.。Like FAM layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HCV RNA feminine gender; Like FAM layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HCV RNA positive.The concrete collection of illustrative plates that detects is shown in Figure 1B.
Be used to detect HIV-1 RNA and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq ID No:17)
Primer
The primer of amplification HCV RNA is HIV-F-L1 (Seq ID No:5) and HIV-F-R1 (Seq ID No:6); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
Fluorescent probe
The nucleotides sequence of the fluorescent probe of HIV is classified HIV-F-P1 (Seq ID No:13) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
The PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acid samples are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 ℃ 10 seconds | 50 ℃ 10 seconds | 65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence gathers FAM and HEX detects wavelength.
The result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, value fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HIV-1 RNA feminine gender; Like FAM layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HIV-1 RNA positive.The concrete collection of illustrative plates that detects is shown in Fig. 1 C.
1.RNA type triple real-time PCR reactions of target nucleic acid
Be used to detect HCV RNA, HIV-1 RNA and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq IDNo:17)
Primer
The primer of amplification HCV RNA is HCV-F-L1 (Seq ID No:3) and HCV-F-R1 (Seq ID No:4); The primer of amplification HCV RNA is HIV-F-L1 (Seq ID No:5) and HIV-F-R1 (Seq ID No:6); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
Fluorescent probe
The nucleotides sequence of the fluorescent probe of HCV is classified HCV-FQ-P1 (Seq ID No:12) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of the fluorescent probe of HIV is classified HIV-F-P1 (Seq ID No:13) as, and its 5 ' end adopts the ROX mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
The PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acid samples are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 ℃ 10 seconds | 50 ℃ 10 seconds | 65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence is gathered FAM, ROX and HEX and is detected wavelength.
The result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HCV RNA feminine gender; Like FAM layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HCV RNA positive.Like ROX layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HIV-1 RNA feminine gender; Like ROX layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like ROX layer Ct value≤45, then be judged to be the HIV-1 RNA positive.Concrete detection collection of illustrative plates is as shown in Figure 2.
Embodiment 3 quadruple real-time PCR reactions
1.DNA/RNA type target nucleic acid quadruple real-time PCR reactions
1.1 be used to detect HBV DNA, HCV RNA, DNA internal reference nucleic acid (Seq ID No:16) and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq ID No:17)
1.1.1 primer
The primer of amplification HBV DNA is HBV-FQ-L1 (Seq ID No:1) and HBV-FQ-R1 (Seq ID No:2); The primer of amplification HCV RNA is HCV-F-L1 (Seq ID No:3) and HCV-F-R1 (Seq ID No:4); The primer of DNA amplification internal reference nucleic acid is IC309L1 (Seq ID No:7) and IC309R1 (Seq ID No:8); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
1.1.2 fluorescent probe
The nucleotides sequence of the fluorescent probe of HBV is classified HBV-FQ-P1 (Seq ID No:11) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of the fluorescent probe of HCV is classified HCV-FQ-P1 (Seq ID No:12) as, and its 5 ' end adopts the TAMRA mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of DNA internal reference nucleic probe is classified IC-FQ-P1 (Seq ID No:14) as, and its 5 ' end adopts the ROX mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
1.1.3 PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acids are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 ℃ 10 seconds | 50 ℃ 10 seconds | 65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence is gathered FAM, ROX, TAMRA and HEX and is detected wavelength.
1.1.4 the result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and ROX layer Ct value＜30, then be judged to be HBV DNA feminine gender; Like FAM layer Ct value＞45 and ROX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HBV DNA positive.Like TAMRA layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HCV RNA feminine gender; Like TAMRA layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like TAMRA layer Ct value≤45, then be judged to be the HCV RNA positive.The concrete collection of illustrative plates that detects is shown in Fig. 3 A.
1.2 be used to detect HBV DNA, HIV-1 RNA, DNA internal reference nucleic acid (Seq ID No:16) and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq ID No:17)
1.2.1 primer
The primer of amplification HBV DNA is HBV-FQ-L1 (Seq ID No:1) and HBV-FQ-R1 (Seq ID No:2); The primer of amplification HIV RNA is HIV-F-L1 (Seq ID No:5) and HIV-F-R1 (Seq ID No:6); The primer of DNA amplification internal reference nucleic acid is IC309L1 (Seq ID No:7) and IC309R1 (Seq ID No:8); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
1.2.2 fluorescent probe
The nucleotides sequence of the fluorescent probe of HBV is classified HBV-FQ-P1 (Seq ID No:11) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of the fluorescent probe of HIV is classified HIV-F-P1 (Seq ID No:13) as, and its 5 ' end adopts the Cy5 mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of DNA internal reference nucleic probe is classified IC-FQ-P1 (Seq ID No:14) as, and its 5 ' end adopts the ROX mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
1.2.3 PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acids are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 ℃ 10 seconds | 50 ℃ 10 seconds | 65 ℃ 45 seconds) | * 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence is gathered FAM, ROX, Cy5 and HEX and is detected wavelength.
1.2.4 the result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and ROX layer Ct value＜30, then be judged to be HBV DNA feminine gender; Like FAM layer Ct value＞45 and ROX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HBV DNA positive.Like Cy5 layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HIV-1 RNA feminine gender; Like Cy5 layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like Cy5 layer Ct value≤45, then be judged to be the HIV-1 RNA positive.The concrete collection of illustrative plates that detects is shown in Fig. 3 B.
Embodiment 4 five heavy real-time PCR reactions
1.DNA/RNA type target nucleic acid five heavy real-time PCR reactions
1.1 be used to detect HBV DNA, HCV RNA, HIV-1 RNA, DNA internal reference nucleic acid (Seq ID No:16) and RNA internal reference nucleic acid (its dna sequence dna of transcribing is with Seq ID No:17)
1.1.1 primer
The primer of amplification HBV DNA is HBV-FQ-L1 (Seq ID No:1) and HBV-FQ-R1 (Seq ID No:2); The primer of amplification HCV RNA is HCV-F-L1 (Seq ID No:3) and HCV-F-R1 (Seq ID No:4); The primer of amplification HIV RNA is HIV-F-L1 (Seq ID No:5) and HIV-F-R1 (Seq ID No:6); The primer of DNA amplification internal reference nucleic acid is IC309L1 (Seq ID No:7) and IC309R1 (Seq ID No:8); The primer of cloning RNA internal reference nucleic acid is IC-F-L2 (Seq ID No:9) and IC-F-R2 (Seq ID No:10).
1.1.2 fluorescent probe
The nucleotides sequence of the fluorescent probe of HBV is classified HBV-FQ-P1 (Seq ID No:11) as, and its 5 ' end adopts the FAM mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of the fluorescent probe of HCV is classified HCV-FQ-P1 (Seq ID No:12) as, and its 5 ' end adopts the TAMRA mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of the fluorescent probe of HIV is classified HIV-F-P1 (Seq ID No:13) as, and its 5 ' end adopts the Cy5 mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of DNA internal reference nucleic probe is classified IC-FQ-P1 (Seq ID No:14) as, and its 5 ' end adopts the ROX mark, the corresponding quenching group of 3 ' end mark; The nucleotides sequence of RNA internal reference nucleic probe is classified IC-F-P2 (Seq ID No:15) as, and its 5 ' end adopts the HEX mark, the corresponding quenching group of 3 ' end mark.
1.1.3 PCR reaction conditions
PCR reaction system TV is 60 μ l, and wherein each component concentrations is respectively: Tris.HCl pH8.3 10mM; KCl50mM; Each 0.2mM of dNTP; Taq archaeal dna polymerase 2U; M-MLV ThermoScript II 25U; RNasin 10U; Each 15pM/ kind of various primers; Each 5pM/ kind of various fluorescent probes; Glycerine 5% (v/v); Each 100 copy number of internal reference nucleic acid; Various target nucleic acids are health ministry visiting central standard sample.
The reaction heat cycling condition is following:
| 42 ℃ 30 minutes | 50 |
94 ℃ 3 minutes | ||
| (94 ℃ 10 seconds | 50 ℃ 10 seconds | 65 ℃ 45 seconds) | 45 circulations |
Use ABI 7500 real-time fluorescence PCR appearance, fluorescence is gathered FAM, ROX, TAMRA, Cy5 and HEX and is detected wavelength.
1.1.4 the result judges
The baseline automatic setting of interpretation of result condition enactment: ABI 7500 luminoscopes, fluorescence threshold are set at 10 times of standard deviation of 3～15 round-robin fluorescent signals.Like FAM layer Ct value＞45 and ROX layer Ct value＜30, then be judged to be HBV DNA feminine gender; Like FAM layer Ct value＞45 and ROX layer Ct value>=30, then need heavily inspection; Like FAM layer Ct value≤45, then be judged to be the HBV DNA positive.Like TAMRA layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HCV RNA feminine gender; Like TAMRA layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like TAMRA layer Ct value≤45, then be judged to be the HCV RNA positive.Like Cy5 layer Ct value＞45 and HEX layer Ct value＜30, then be judged to be HIV-1 RNA feminine gender; Like Cy5 layer Ct value＞45 and HEX layer Ct value>=30, then need heavily inspection; Like Cy5 layer Ct value≤45, then be judged to be the HIV-1 RNA positive.Concrete detection collection of illustrative plates is as shown in Figure 4.
Sequence table
< 110>Shanghai Haoyuan Biotechnology Co., Ltd.
< 120>the PCR detection method and the test kit thereof of multiple-target nucleic acid in the single tube
<160>17
<210>1
<211>17
<212>DNA
< 213>artificial sequence
<400>1
tgtcctggtt?atcgctg 17
<210>2
<211>20
<212>DNA
< 213>artificial sequence
<400>2
cagaagaacc?aacaagaaga 20
<210>3
<211>20
<212>DNA
< 213>artificial sequence
<400>3
ttcacgcaga?aagcgtctag 20
<210>4
<211>22
<212>DNA
< 213>artificial sequence
<400>4
aattccggtg?tactcaccgg?tt 22
<210>5
<211>30
<212>DNA
< 213>artificial sequence
<400>5
agtgggggga?catcaagcag?ccatgcaaat 30
<210>6
<211>28
<212>DNA
< 213>artificial sequence
<400>6
tactagtagt?tcctgctatg?tcacttcc 28
<210>7
<211>23
<212>DNA
< 213>artificial sequence
<400>7
tcactgaagc?gggagggac?tggc 23
<210>8
<211>23
<212>DNA
< 213>artificial sequence
<400>8
gcaggtagcc?ggatcaagcg?tat 23
<210>9
<211>22
<212>DNA
< 213>artificial sequence
<400>9
cttcagcaat?atcacgggt?agc 23
<210>10
<211>23
<212>DNA
< 213>artificial sequence
<400>10
gatgcctgct?tgccgaatat?cat 23
<210>11
<211>21
<212>DNA
< 213>artificial sequence
<400>11
tgtgtctgcg?gcgttttatc?a 21
<210>12
<211>23
<212>DNA
< 213>artificial sequence
<400>12
atggcgttag?tatgagtgtc?gtg 23
<210>13
<211>28
<212>DNA
< 213>artificial sequence
<400>13
accatcaatg?aggaagctgc?agaatggg 28
<210>14
<211>20
<212>DNA
< 213>artificial sequence
<400>14
ccgccgcatt?gcatcagcca?tgatg 25
<210>15
<211>21
<212>DNA
< 213>artificial sequence
<400>15
cctgatagcg?gtccgccaca?ccc 23
<210>16
<211>142
<212>DNA
< 213>artificial sequence
<400>16
gcaggtagcc?ggatcaagcg?tatgcagccg?ccgcattgca?tcagccatga?tggatacttt 60
ctcggcagga?gcaaggtgag?atgacaggag?atcctgcccc?ggcacttcgc?ccaatagcag 120
ccagtcccttc?ccgcttcagt?ga 142
<210>17
<211>124
<212>RNA
< 213>artificial sequence
<400>17
cuucagcaau?aucacgggu?agccaacgcu?auguccugau?agcgguccgc?cacacccagc 60
cggccacagu?cgaugaaucc?agaaaagcgg?ccauuuucca?ccaugauauu?cggcaagcag 120
gcauc 124
Claims (9)
1. the real-time PCR method of multiple target nucleic acid in the multiple test sample in single PCR reaction vessel of non-diagnostic purpose, every kind of target nucleic acid is respectively HCV RNA, HIV RNA or HBV DNA, it comprises,
(1) in said single PCR reaction vessel, mix ThermoScript II, sample, archaeal dna polymerase, dNTP, be selected from SEQ ID NO:1 and 2,3 and 4,5 and 6 multiple target nucleic acid primer to, be selected from SEQ ID NO:11,12 and 13 multiple target nucleic acid probes, be selected from SEQ ID NO:16 and 17 internal reference nucleic acid, be selected from SEQ ID NO:7 and 8,9 and 10 internal reference nucleic acid primer to, be selected from the internal reference nucleic probe of SEQ ID NO:14 and 15; Wherein aforementioned various probe marks have fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different;
(2) carry out the PCR reaction, and detect the fluorescence of different wave length in real time;
(3) the Ct value that calculates according to the fluoroscopic examination result has judged whether that one or more target nucleic acids are present in the sample.
2. the described method of claim 1, wherein said internal reference nucleic acid is packaged in the virus-like particle.
3. the described method of claim 1, wherein various probe marks have different fluorophors.
4. the described method of claim 1, wherein each round-robin condition of PCR reaction is 94 ℃ 10 seconds, 50 ℃ 10 seconds and 65 ℃ 45 seconds.
5. the described method of claim 1, wherein sample is the sample of handling through the magnetic bead extraction method.
6. the described method of claim 1, Ct value≤45 that calculate of a kind of fluoroscopic examination result of target nucleic acid wherein, then this target nucleic acid is present in the sample; Ct value＞45 that a kind of fluoroscopic examination result of target nucleic acid calculates, and Ct value＜30 that calculate of the fluoroscopic examination result of corresponding internal reference nucleic acid, then this target nucleic acid is not present in the sample.
7. detection kit; Said detection kit comprises; ThermoScript II, archaeal dna polymerase, dNTP, be selected from SEQ ID NO:1 and 2,3 and 4,5 and 6 multiple target nucleic acid primer to, be selected from SEQ ID NO:11,12 and 13 multiple target nucleic acid probes, be selected from SEQ ID NO:16 and 17 internal reference nucleic acid, be selected from SEQ ID NO:7 and 8,9 and 10 internal reference nucleic acid primer to, be selected from the internal reference nucleic probe of SEQ ID NO:14 and 15; Wherein aforementioned various probe marks have fluorophor and quenching group, and the fluoroscopic examination wavelength of the fluorophor of various probe marks is different.
8. the described a kind of detection kit of claim 7, wherein said internal reference nucleic acid is packaged in the virus-like particle.
9. the described a kind of detection kit of claim 7, wherein said detection kit comprises the required reagent of magnetic bead extraction method.
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| GB201503775D0 (en) * | 2015-03-05 | 2015-04-22 | Bg Res Ltd | Multiplexed detection of nucleic acid targets directly from samples containing blood |
| CA2983428C (en) * | 2015-06-04 | 2021-12-07 | Mizuho Medy Co., Ltd. | Kit for together detecting multiple target nucleic acids differing from each other and detection method using the same |
| CN105624311A (en) * | 2016-03-08 | 2016-06-01 | 湖南圣湘生物科技有限公司 | Real-time fluorescent PCR detection method and kit of multiple target nucleic acid in single tube |
| CN105695631B (en) * | 2016-03-18 | 2019-04-23 | 广东出入境检验检疫局检验检疫技术中心 | Human immunodeficiency virus, hepatitis type B virus, Hepatitis C Virus Quick joint inspection kit and its preparation and application |
| CN107287348A (en) * | 2017-06-19 | 2017-10-24 | 广州和实生物技术有限公司 | A kind of blood sieves three polychrome single tube detection kits |
| CN109182600B (en) * | 2018-09-18 | 2021-10-22 | 中国科学院合肥物质科学研究院 | Fluorescent quantitative PCR kit for synchronously detecting hepatitis B virus, hepatitis C virus and human immunodeficiency virus type 1 |
| CN109576397B (en) * | 2018-12-28 | 2022-10-14 | 广州达安基因股份有限公司 | Human immunodeficiency virus type 1 nucleic acid quantitative detection kit |
| 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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