CN102816872A - Fluorescence quantitative polymerase chain reaction (PCR) detection kit for hepatitis B virus (HBV) - Google Patents
Fluorescence quantitative polymerase chain reaction (PCR) detection kit for hepatitis B virus (HBV) Download PDFInfo
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- CN102816872A CN102816872A CN2012103331473A CN201210333147A CN102816872A CN 102816872 A CN102816872 A CN 102816872A CN 2012103331473 A CN2012103331473 A CN 2012103331473A CN 201210333147 A CN201210333147 A CN 201210333147A CN 102816872 A CN102816872 A CN 102816872A
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Abstract
The invention relates to a fluorescence quantitative PCR detection kit for HBV. The kit comprises a PCR master mix, a fluorescence quantitative reaction solution and a positive control sample. The fluorescence quantitative reaction solution refers to a reaction system comprising a forward primer, a reverse primer and a locked nucleotide probe which are used for detecting HBV DNA, wherein the sequence of the forward primer is SEQ ID NO:1, the sequence of the reverse primer is SEQ ID NO:2, the sequence of the Taqman probe is SEQ ID NO:3, the 5'end of the probe is marked with FAM, the 3'end of the probe is marked with a minor groove binder (MGB), and the positive control sample A comprises DNA plasmid of HBV genome fragments. The detection kit is suitable for quantitative detection of HBV nucleic acid in various tissues, cells, blood and other body liquid.
Description
Technical field
The invention belongs to biology field, relate to a kind of detection hbv nucleic acid PCR kit for fluorescence quantitative, be applicable to the detection by quantitative of the hbv nucleic acid in various tissues, cell, blood and other body fluid.
Background technology
(hepatitis B virus HBV) infects and not only to cause acute and chronic hepatitis hepatitis B virus, and closely related with hepatic fibrosis, hepatocellular carcinoma, is the public health problem of a serious harm whole world human health.About 2,000,000,000 people in the whole world have been proved to be hepatitis B infected, and 3.5 hundred million people are chronic infection person, and according to the World Health Organization, in preceding 10 the disease causes of the death in the whole world, hepatitis B accounts for the 9th.China belongs to HBV infection district occurred frequently in the world, and about 1.3 hundred million people in the whole nation are HBV carrier, and chronic hepatitis patient 2,300 ten thousand accounts for global HBV surface antigen (hepatitis B virus surface antigen, HBsAg) nearly 50% of total carrying rate; 60% people received the infection of HBV, in some crowd even up to 79%; 8% ~ 10% people is the HBsAg carrier.In addition, have approximately among the patient of trouble chronic hepatitis to be chronic hepatitis B patient more than 80%, (hepatocellular carcinoma, relative risk HCC) increases by 300 times at least and receive the chronically infected crowd of HBV to suffer from primary hepatocellular carcinoma.According to World Health Organization's nineteen eighty-three report, about 80% is all relevant with the HBV chronic infection among the global primary HCC.Annual China has 50,000,000,000 yuan (do not comprise the healthcare products expense, interfere with one's studies, obtain employment and indirect loss that work etc. causes) approximately in expense aspect the direct treatment of viral hepatitis.It is most important that early diagnosis HBV index of correlation spreads, establishes correct regimen to the control hepatitis B.
Hepatitis B virus belongs to has a liking for hepatovirus family, is one of animal DNA virus of known minimum.The HBV genome structure is unique, is made up of its two chain nucleic acid different in size incomplete circular double stranded DNA.Four open reading frames (ORF) are arranged, i.e. S district, C district, P district and X district, the HBsAg that encodes respectively, HBcAg, HBeAg, DNA polymerase and X protein on the long-chain of HBV-DNA.It is the most frequently used index of diagnosis of hepatitis b that hepatitis B virus blood serum designated object (HBV-M) detects; At present clinical the most frequently used " two double " refer to HBsAg, HBeAg, HBcAb, HBsAb, HBeAb; The immunological status that main reflection human body infects hepatitis B virus (HBV); But can not directly reflect HBV in the intravital situation of duplicating of patient, more can not be as judging whether the patient has communicable direct evidence.Because HBV the infected's different times can have multiple different serologic marker pattern, for the different clinical meanings of these patterns, the non-infective disease specialist is difficult to accurately grasp.Because HBV is a dna virus; Therefore the detection of HBV DNA is to weigh hepatitis B the most accurate communicable index; The HBV DNA positive promptly reflects has duplicating of complete virion; Be to judge that the patient has direct and the most reliable communicable index, simultaneously, HBV DNA also can be used as an extremely significant clinical indices judging the HBV curative effect of medication.Mainly adopt ELISA that the immune marker on HBV surface is detected with quantitative now clinically. but the specificity of enzyme linked immunological is not high. and sensitivity is relatively low. and false negative rate is high.After the nineties, round pcr and biochip technology have been used in the detection of hepatitis B virus, have improved the accuracy that detects greatly; Valve when having shortened analysis; Realized detection by quantitative, can judge the HBV virus situation of duplicating in vivo accurately, but also there have been various defectives in these detection techniques HBV DNA; And cost is higher relatively, is unfavorable for popularizing.Therefore, exploitation fast, the technology of responsive and special detection HBV DNA is the developing direction of following HBV etiology detection technique.
HBV DNA quantitative detecting method commonly used at present has technology such as hybrid method, PCR (polymerase chain reaction), COBAS Amplicor technology and bDNA.Using COBAS Amplicor to detect HBV DNA is the gold standard of internationally recognized PCR quantivative approach, has good accuracy and repeatability.Its principal feature is: owing to adopted internal reference, can monitor the extracting of viral nucleic acid, the efficient of amplification effectively, can eliminate the influence of interfering factors in the reaction system, the content of target nucleic acid in the time of therefore can reflecting the sample original state more objectively.But, be difficult in clinical labororatory and promote the use of because its matched reagent is extremely expensive.BDNA is higher at the lower limit of HBV DNA quantitatively determined neutral line scope, thereby its clinical value is also limited.
The base that hybrid method is based on the nucleic acid molecule composition can combine through pairing is complementary.Have affinity tag DNA or RNA fragment can with complementary nucleotide sequence specific combination, this fragment is called nucleic probe.The nucleic probe of HBV has two kinds of dna probe and rna probes.The principal feature of nucleic probe method is that susceptibility, specificity are all stronger, but detection sensitivity is low.
The PCR method include the nido polymerase chain reaction (nested PCR, nPCR), multiplex PCR (multiplex PCR, mPCR), competitive PCR (competitive PCR, several different methods such as cPCR).NPCR is a qualitative checking method, can not direct quantitative, and its product need qualitatively judge through gel electrophoresis; The mPCR method is saved time and reagent, can more diagnostic messages more accurately be provided for clinical, but need many primers of design, and product also need be judged through carrying out gel electrophoresis; The advantage of cPCR is to control the difference that the differential responses pipe is asked amplification efficiency effectively; And the accommodation of target gene concentration is bigger; But this method need be carried out the isotope probe of gel electrophoresis, Southern blot and mark and carried out quantitative analysis in the entire operation process; Though susceptibility is high, testing conditions is not easy to stdn.
The real-time fluorescence quantitative PCR of up-to-date appearance combines nucleic acid amplification, hybridization, spectroscopic analysis and real-time detection technique; Detect the PCR product by means of fluorescent signal, having solved conventional P CR technology can not be quantitatively and the amplified production pollution problems, has avoided the pollution in the common quantitative PCR operating process; Make it easy and simple to handle, quick; The result is accurate, has become the strong tool of quantitative gene expression, has characteristics such as susceptibility and specificity height, good reproducibility and quantitative scope are wide.Detection by quantitative requires that detection method is responsive, special, accurate, accurate, good reproducibility, linearity range are wide, and between each genotype indifference.And full automatic quantitative fluorescent PCR becomes the most promising quantitative measurement technology because of its more excellent accuracy, tolerance range and repeatability.
Real-time fluorescence quantitative PCR technology (real-time fluorescent quantitative PCR; FQ-PCR) released by U.S. Applied Biosystems company in 1996; It is a kind of specific fluorescent probe that when pcr amplification, when adding a pair of primer, adds; This probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the reporter group fluorescent signal emitted was absorbed by quenching group; When just beginning, probe is combined on the arbitrary strand of DNA; During pcr amplification; 5 ' end-3 ' the end 5 prime excision enzyme activity of Taq enzyme is cut degraded with probe enzyme; The report fluorophor is separated with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal, DNA chain of promptly every amplification; Just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form fully synchronously.This technology not only realized to dna profiling quantitatively; And have highly sensitive, specificity and safety is stronger, can realize characteristics such as multiple reaction, level of automation height, nonstaining property, tool real-time and accuracy, be widely used in fields such as molecular biology research and medical research at present.
Summary of the invention
The object of the present invention is to provide the fluorescent quantificationally PCR detecting kit of quick, the high sensitive detection serum of a kind of ability, blood plasma HBV DNA.
Described test kit comprises: fluorescent quantitation reaction solution premixed liquid (PCR Master Mix), fluorescent quantitation reaction solution, positive control sample.
The present invention adopts lock nucleic acid (locked nucleic acid; LNA) the synthetic pulsating lock nucleotide sequence of the HBV gene nucleic acid probe that covers of technology; The combined with fluorescent quantitative PCR technology is developed a kind of quick, super-sensitive detection HBV gene by fluorescence quantitative PCR test kit.Test kit of the present invention comprises fluorescent quantitation reaction solution premixed liquid (PCR Master Mix), fluorescent quantitation reaction solution, positive control sample.
The fluorescent quantitation reaction solution contains positive and negative primer and fluorescently-labeled HBV-DNA lock nucleotide probe.
Forward primer (HBV-Forward Primer) sequence is:
SEQ?ID?NO:1（5’-CCTATGGGAGTGGGCCTCA-3’）；
Reverse primer (HBV-Reverse Primer) sequence is:
SEQ?ID?NO:2（5’-CATCCATATAACTGAAAGCCAAACAGT-3’）；
Fluorescent probe (HBV-Taqman-Probe) sequence is:
SEQ ID NO:3 (5 '-FAM-CTAGTGCCATTTGTTC-MGB-3 ') its 5 ' end flag F AM (fluorescence report group), 3 ' end mark MGB (fluorescent quenching group).
Positive control sample A: comprise the HBV genomic fragment DNA plasmid that detects.
The principle of HBV thymus nucleic acid fluorescent quantificationally PCR detecting kit is: adopt fluorescence quantifying PCR method that HBV is carried out detection by quantitative.A pair of to the special design of HBV gene order TaqMan probe and primer; When running into the HBV specific gene sequence; The TaqMan probe can combine with it fully, and during pcr amplification, the fluorescence report group sends fluorescence because of enzymolysis separates; Can detect the accumulation of corresponding fluorescent signal in real time, thereby realize detecting the purpose of HBV content.
The TaqMan-MGB probe that the present invention adopts is compared with traditional T aqMan-TAMRA probe, and the MGB probe has the TM of raising value, make the contraction in length of probe, and the TM value difference that improves between pairing and non-matching template is different; Improve SNR, make experimental result more accurate, resolving power is higher; More simplify experiment, MGB probe optimum experimental step is simple, and the stability of hybridization improves greatly, and repeatability such as improves greatly at advantage.
Two ends provided by the invention all indicate the specificity fluorescent probe of fluorescence radiation group; When probe is complete; Two groups on space structure the distance each other near; 5 ' end fluorescence of sending of reporter group is because FRET (FRET) and by the group cancellation of going out of 3 ' end quenching, so there is not the variation of fluorescent signal in the system.In case and it and the combining of template specificity; Its binding site is between two primers, and along with the extension of primer, the Taq archaeal dna polymerase runs into the probe that combines with template in the chain extension process; Its 5 '-3 ' 5 prime excision enzyme activity will cut off probe; The fluorescence report group has so just destroyed the FRET between two fluorophors away from the fluorescent quenching group, and the photofluorometer that the fluorescence that reporter group discharged just can be built in the detection by quantitative appearance detects.PCR is every through a circulation, and fluorescent signal is also the same with the purpose fragment, and the process that has sync index to increase, the power of fluorescent signal have just been represented what of copy number of template DNA.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sample.
Compare with the test kit of immunology detection HBV virus, and compare with the test kit of conventional fluorescence quantitative PCR detection HBV-DNA gene, test kit of the present invention has following advantage:
1. highly sensitive: sensitivity reaches 10 every microlitre test sample of gene copy number, and 10 times are superior to common fluorescence quantitative PCR method;
Detection time short (from sample inspect by ready samples to obtain a result can be at 12 hours with interior completion);
3. operating process is simple, and from PCR reaction beginning, is exactly in the system of sealing, to accomplish amplification and The real time measure, greatly reduces contamination of heavy, has therefore also just reduced the probability of result error;
4. present technique is lower to the specification of quality that specimen dna obtains, and no matter is serum, blood plasma, paraffin organization or flesh tissue, can both obtain the ideal detected result;
5. the sample size that detects is big, and once experiment can detect 384 examples at most;
6. safety: do not comprise hazardous and noxious substances in the whole system, operator and environment are not had harm.
Test kit of the present invention is the HBV DNA fluorescent quantificationally PCR detecting kit of a new generation; Utilize present state-of-the-art lock nucleic acid technique; Make the more common quantitative PCR of detection by quantitative sensitivity of HBV DNA improve 10-100 doubly; On the one hand, can be used for that diagnosis of hepatitis b treatment, prognosis are judged, clinical application such as recurrence and transfer behind the monitoring liver cancer treatment; Can estimate the degree that HBV infects in the patient body through HBV dna level in the detection of dynamic patient body on the other hand, thereby estimate patient's immunizing power change dynamics.
Description of drawings
Fig. 1 is that employing test kit detectable level of the present invention is 10
4-10
7The quantitative fluorescent PCR graphic representation of copy/microlitre HBV DNA standard substance.Among the figure, from left to right, be respectively 10
7Copy/microlitre, 10
6Copy/microlitre, 10
5Copy/microlitre, 10
4The HCV RNA fluorescent quantitative PCR curve of copy/microlitre.
Fig. 2 is the FQ-PCR graphic representation of test kit of the present invention test section positive sample.
Above-mentioned accompanying drawing is the original detection figure that detecting instrument carries software, and the English implication of X-coordinate is " a PCR cycle number " among the figure, and the English implication of ordinate zou is " fluorescent value of amplified reaction ".
Embodiment
Embodiment 1: test kit of the present invention is for the sensitivity of standard model
Adopt fluorescent quantificationally PCR detecting kit detectable level of the present invention to be respectively 10
4Copy/microlitre, 10
5Copy/microlitre, 10
6Copy/microlitre, 10
7The HBV DNA standard substance of copy/microlitre.The standard substance of this experiment are precious biotechnology (Dalian) ltd synthetic DNA.
Test kit of the present invention contain custom-designed can specific detection fluorescent probe and the primer of HBV DNA a pair of:
Forward primer (HBV-Forward Primer):
5’-CCTATGGGAGTGGGCCTCA-3’
Reverse primer (HBV-Reverse Primer):
5’-CATCCATATAACTGAAAGCCAAACAGT-3’
Fluorescent probe (HBV-Taqman-Probe):
5’-FAM-CTAGTGCCATTTGTTC-MGB-3’
Optimizing reaction system is carried out the detection of FQ-PCR then; Fluorescent quantitation is reflected on the ABI7900 detector and carries out, and reaction system is 15 μ l, each 0.15 μ l (20 μ Μ) of positive and negative primer; Each 0.2 μ l (20 μ Μ) of probe; Template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.
Pcr amplification reaction program: 95 ℃ of preparatory sex change 30 seconds; And by 95 ℃ 5 seconds, 60 ℃ 33 seconds, amplified reaction 40 times circulation.
The result is: from left to right, be respectively 10
7Copy/microlitre, 10
6Copy/microlitre, 10
5Copy/microlitre, 10
4The HBV RNA fluorescent quantitative PCR curve of copy/microlitre.
This experiment confirms that tentatively test kit of the present invention possesses higher susceptibility and specificity, and false positive is low.
Embodiment 2: test kit of the present invention is for the sensitivity of clinical sample
Adopt fluorescent quantificationally PCR detecting kit of the present invention that 30 routine clinical samples (deriving from the patients serum that hepatitis B virus is contained in tumour hospital of Zhongshan University) are detected.
Adopt fluorescent quantificationally PCR detecting kit of the present invention that 30 routine clinical samples are detected.
Test kit of the present invention contain custom-designed can specific detection fluorescent probe and the primer of HBV DNA a pair of:
Forward primer (HBV-Forward Primer):
5’-CCTATGGGAGTGGGCCTCA-3’
Reverse primer (HBV-Reverse Primer):
5’-CATCCATATAACTGAAAGCCAAACAGT-3’
Fluorescent probe (HBV-Taqman-Probe):
5’-FAM-CTAGTGCCATTTGTTC-MGB-3’
Optimizing reaction system is carried out the detection of FQ-PCR then; Fluorescent quantitation is reflected on the ABI7900 detector and carries out, and reaction system is 15 μ l, each 0.15 μ l (20 μ Μ) of positive and negative primer; Each 0.2 μ l (20 μ Μ) of probe; Template DNA 2.5 μ l (100-300ng/ μ l), 2*Taqman universal PCR Master Mix (available from U.S. applying biological company) 7.5 μ l, ddH
2O 4.3 μ l.PCR reaction conditions: 95 ℃ of preparatory sex change 30 seconds; And by 95 ℃ 5 seconds, 60 ℃ 33 seconds, amplified reaction 40 times circulation.
The result is: numbering is respectively the corresponding hole of detecting of 2,7,13,24,25,28 sample has amplification curve, and detected result is positive, and fluorescence quantitative PCR detection result is as shown in Figure 2, and the detected result that shows this test kit has high specificity and safety.
In sum, test kit of the present invention is not only fast and efficiently, and its result's interpretation is very clear and definite, directly perceived, reliable results, special.
Claims (3)
1. fluorescence quantitative PCR detection kit for hepatitis B virus, comprising: fluorescent quantitation reaction solution premixed liquid, fluorescent quantitation reaction solution, positive control sample is characterized in that:
Said fluorescent quantitation reaction solution comprises the positive and negative primer and the reaction system of locking nucleotide probe that contains detection HBV DNA; Wherein, The forward primer sequence is SEQ ID NO:1, and the reverse primer sequence is SEQ ID NO:2, and the fluorescent probe sequence is SEQ ID NO:3; Its 5 ' end mark fluorescent reporter group, 3 ' end mark fluorescent quenching group;
Positive control sample A: comprise the HBV genomic fragment DNA plasmid that detects.
2. fluorescence quantitative PCR detection kit for hepatitis B virus according to claim 1 is characterized in that: the fluorescence report group of 5 of said fluorescent probe ' end mark is FAM.
3. fluorescence quantitative PCR detection kit for hepatitis B virus according to claim 1 is characterized in that: the fluorescent quenching group of 3 of said fluorescent probe ' end mark is MGB.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109694926A (en) * | 2018-12-29 | 2019-04-30 | 中山大学达安基因股份有限公司 | A kind of method and kit of quantitative detection HBV nucleic acid |
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| CN101812533A (en) * | 2009-02-24 | 2010-08-25 | 江苏默乐生物科技有限公司 | Primer and probe for fluorescent quantitative PCR detection of hepatitis B viruses (HBV) |
| CN102146487A (en) * | 2011-04-12 | 2011-08-10 | 武汉百泰基因工程有限公司 | Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid |
| CN102417936A (en) * | 2011-12-13 | 2012-04-18 | 泰普生物科学（中国）有限公司 | Polymerase chain reaction (PCR) fluorescence quantitative detection kit and method for hepatitis B viruses (HBV) |
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Patent Citations (4)
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| EP1152063A1 (en) * | 2000-05-03 | 2001-11-07 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Method of diagnosing HBV infection stages |
| CN101812533A (en) * | 2009-02-24 | 2010-08-25 | 江苏默乐生物科技有限公司 | Primer and probe for fluorescent quantitative PCR detection of hepatitis B viruses (HBV) |
| CN102146487A (en) * | 2011-04-12 | 2011-08-10 | 武汉百泰基因工程有限公司 | Quantitative detection kit, detection method, primer and probe for hepatitis B virus nucleic acid |
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| CN109694926A (en) * | 2018-12-29 | 2019-04-30 | 中山大学达安基因股份有限公司 | A kind of method and kit of quantitative detection HBV nucleic acid |
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Effective date of registration: 20160728 Address after: 241000 Anhui city of Wuhu Province Economic and Technological Development Zone Branch Valley Park No. 9 Building Applicant after: ANHUI DAJIAN MEDICAL TECHNOLOGY CO., LTD. Address before: 510006 Guangdong city of Guangzhou province Panyu District Xiaoguwei Street Outer Ring Road No. 280 Building No. 401 room a department of Guangdong Pharmaceutical University Applicant before: Guangzhou Dajian Biotechnology Co., Ltd. |
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Application publication date: 20121212 |