CN102417936B

CN102417936B - Polymerase chain reaction (PCR) fluorescence quantitative detection kit and method for hepatitis B viruses (HBV)

Info

Publication number: CN102417936B
Application number: CN 201110417476
Authority: CN
Inventors: 何东华; 黄发平
Original assignee: TRIPLEX INTERNATIONAL BIOSCIENCES (CHINA) CO Ltd
Current assignee: TRIPLEX INTERNATIONAL BIOSCIENCES (CHINA) CO Ltd
Priority date: 2011-12-13
Filing date: 2011-12-13
Publication date: 2013-09-18
Anticipated expiration: 2031-12-13
Also published as: CN102417936A

Abstract

The invention relates to a disease pathogen gene detection technology, in particular to quantitative detection of hepatitis B viruses (HBV) in a serum or plasma sample. According to the technical scheme, the polymerase chain reaction (PCR) fluorescence quantitative detection kit for the HBV comprises a PCR reaction solution; the PCR reaction solution contains primers A and a fluorescence probe A;the primers A are divided into an upstream primer A and a downstream primer A; and the kit also comprises DNA polymerase, UNG enzyme, a strong positive quality control, a weak positive quality control, a negative quality control, a positive quantitative reference and a DNA extract. According to the PCR fluorescence quantitative detection kit and a PCR fluorescence quantitative detection method, the detection sensitivity is higher by using a Taqman core technology platform and an arabidopsis thaliana internal control system. Compared with the conventional product, the kit has the advantage of improving accuracy, specificity, repeatability, stability, sensitivity and precision.

Description

A kind of hepatitis B virus PCR fluorescence quantitative detection kit and method thereof

Technical field

The present invention relates to a kind of disease pathogen technique of gene detection, relate in particular to hepatitis B virus in detection by quantitative serum or the plasma sample (HBV) PCR fluorescence quantitative detection kit and the method thereof of being applicable to.

Background technology

(hepatitis B virus is one of the hepatitis virus of serious harm human health HBV) to hepatitis B virus, is important hepatic diseases virulence factor.There are 2,000,000,000 people to infect HBV in the world, whole world chronic HBV infection patient has 3.8 hundred million at least at present, wherein Chronic HBV carrier 75% is distributed in South East Asia and time area, the Sahara, and China has just accounted for 1.4 hundred million, it is the chronic viral hepatitis B patient that 2,000 ten thousand people are wherein arranged, and the annual people who dies that therefore dies of illness has nearly 500,000.Estimate that the whole world has 100-200 ten thousand people directly to die from the HBV persistent infection every year.

HBV belongs to Hepadnaviridae, it is a kind of dna virus, its genome is made up of the dsdna segment of long 3.2kb, have four opening code-reading frames (Open Reading Frame, ORF): S, C, P, X, their at least 8 different albumen of encoding: surface antigen protein, DNA polymerase protein, cAg albumen, X protein etc.HBV has the feature of virus replication output height (1012-13 virion/sky) and mutation rate height (1010-11 point mutation/sky), high sudden change is because height copies, especially owing to genome duplication, need be transcribed into the RNA intermediate earlier, but polysaccharase lacks due to the correct functioning.According to the full gene nucleotide series heterology of HBV greater than 8% or S gene regions nucleotide sequence heterology greater than 4.1%, HBV can be divided into A, B, C, D, E, F, G, a H8 genotype.

Relevant clinical and laboratory diagnostic method:

The technique means of laboratory detection hepatitis b virus marker is more, wherein the most normal employing enzyme immunosorbent adsorption test (ELISA method) detects serum of hepatitis B and learns mark (HBV-M), it has quick, inexpensive, easy characteristics, can be used for the examination in enormous quantities of hepatitis B.But because this method can only qualitative analysis, and hepatitis B two double results also have 10 surplus kind, often cause the clinical diagnosis difficulty.Previously Southern commonly used and spot hybridization detect HBV DNA in the domestic and foreign literature, and these two kinds of required sample sizes of method are bigger, are difficult to accurately quantitatively.

Hepatitis B two double detections are most representative serology detection methods, and it is fast and convenient to be suitable for the hepatitis B examination.But the defective of these class methods is to be indirect antigen-antibody inspection, can not itself detect virus, also can't provide essential reference for the judgement that chronic HBV infection patient's treatment and the course of disease are made progress.

Genotype Auele Specific Primer PCR method usually need be by the gel electrophoresis sentence read result, this be subjected to much can not the certainty factor restriction interpretation method as a result avoid being applied to clinical detection always, so this method is limited to the scientific research field at present more.

RFLP complex operation and poor repeatability, higher to testing staff's technical requirements, make it to be used widely clinically.

The gene sequencing method is method the most accurately and reliably, but the equipment that order-checking needs and technical threshold make order-checking can only be confined to some unit with certain strength carries out, country's associated mechanisms also can't carry out quality control to the sequencing result of different detected objects, it is higher to add the order-checking expense, makes gene sequencing can't be applied to clinical detection at all.

Summary of the invention

The object of the invention provides a kind of test kit that is applicable to the rapid detection of hepatitis B virus in the clinical sample, can be used for the monitoring of hepatitis b virus infected auxiliary diagnosis and curative effect.

For achieving the above object, technical scheme of the present invention comprises the PCR reaction solution for a kind of hepatitis B virus PCR fluorescence quantitative detection kit is provided, and described PCR reaction solution contains primer A and fluorescent probe A; Described primer is divided into upstream primer A and downstream primer A:

Upstream primer A sequence is as 5 '-TCTTCTTGTTGGTTCTTCTGGACT-3 ';

Downstream primer A sequence is as 5 '-AAACATAGAGGTTCCTTGAGCAGG-3 ';

Fluorescent probe A sequence is as 5 '-CAAGGTATGTTGCCCGTTTGTCCTCTA-3 '.

As the further setting of the present invention, described test kit also comprises DNA extraction liquid, archaeal dna polymerase, UNG enzyme, strong positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference product.

As the further setting of the present invention, introduced an artificial constructed interior reference system and be used for avoiding detected result false negative, interior reference system to comprise confidential reference items probe, primer and confidential reference items DNA.

As the further setting of the present invention, described interior reference system is reference system in the Arabidopis thaliana, and described interior reference system comprises the upstream primer sequence B: SEQ ID NO.4, downstream primer sequence B: SEQ ID NO.5 and fluorescent probe sequence B: SEQ ID NO.6.

Another technical scheme of the present invention is for providing a kind of hepatitis B virus PCR fluorescence quantitative detecting method, wherein the pcr amplification the primer is: upstream primer A sequence such as SEQ ID NO.1, downstream primer A sequence such as SEQ ID NO.2, fluorescent probe sequence A such as SEQ ID NO.3.

As the further setting of aforesaid method, 5 ' the end flag F AM fluorophor of described fluorescent probe A, 3 ' end mark TAMRA quenching group.

Hepatitis B virus PCR fluorescence quantitative detection kit of the present invention and method have been utilized reference system in Taqman core technology platform and the Arabidopis thaliana, and can effectively remove the influence of the inhibition that suppresses pcr amplification, and it is quantitatively more accurate to make.Compare with the simple PCR detection by quantitative in laboratory, this product accuracy height, high specificity, and in repeatability, stability, sensitivity and precision very big improvement has been arranged, and improve, show that it possesses following performance:

(1) accuracy: 9 parts of positive reference material P1-P9 detection coincidence rates are 100% in the national standard product, and coincidence rate is 100%.

(2) specificity: detection specificity reference material result is all negative.

(3) sensitivity: the minimum detectability of energy stable detection CT-DNA is 5.0 * 10 ²Copies/mL.

(4) precision: detect the internal control reference product P1 of enterprise and P2, each sample repeats 10 times, calculates batch interpolation and the equal CV value of difference between batch≤10%.

Description of drawings

Fig. 1: positive reference material detected result figure;

Fig. 2: positive reference material interior with reference to detected result figure;

Fig. 3: be specificity reference material detected result figure;

Fig. 4: in the specificity reference material with reference to fluoroscopic examination figure as a result

Fig. 5: be sensitivity reference material detected result figure;

Fig. 6: positive quantitatively reference product detected result figure;

Fig. 7: be the typical curve made from Fig. 4 result;

Fig. 8: precision detected result figure;

Fig. 9: strong positive and weak positive quality control product detected result figure.

Embodiment

This test kit adopts polymerase chain reaction (PCR) and fluorescence labeling probe technology, the peculiar conserved sequence of hepatitis B virus in the rapid detection clinical sample, shown in conserved sequence shown in SEQ ID NO.7, thereby judge the existence of hepatitis B virus.

Composition and the configuration of embodiment 1 test kit

One, main raw material(s) source and preparation method:

Primer and the probe sequence of this test kit are as follows:

Upstream primer A is shown in SEQ ID NO.1: 5 '-TCTTCTTGTTGGTTCTTCTGGACT-3 ';

Downstream primer A is shown in SEQ ID NO.2: 5 '-AAACATAGAGGTTCCTTGAGCAGG-3 ';

Fluorescent probe A is shown in SEQ ID NO.3:

5′-FAM-CAAGGTATGTTGCCCGTTTGTCCTCTA-TAMRA-3′。

Interior reference (Suc) primer and the probe sequence of this test kit are as follows:

Upstream primer B is shown in SEQ ID NO.4: 5 '-TCATCGTCGCTGGAGCTGGTT-3 ';

Downstream primer B is shown in SEQ ID NO.5: 5 '-CGGCGGTTTGTCAAGCTGAT-3 ';

Fluorescent probe B is shown in SEQ ID NO.6:

5′-HEX-CTTCTTATAGTCACTGCACTAAACTGGAT-TAMRA-3′。

Interior with reference to principle:

Test kit is provided with interior reference, should be interior with reference to being the plasmid that contains arabidopsis gene (SUC gene), the SUC gene is one section gene that has nothing to do with people and hepatitis B virus, and whether reference has the PCR inhibition to exist to detect in each PCR reaction in therefore can be used as, thereby guarantees PCR result's credibility.When interior be when sun with reference to the result, expression PCR reaction system and operation are normal; Therefore when the goal gene result is the moon, interiorly just seem very important with reference to the result for sun; But when the goal gene result is when sun, the amplification curve of interior reference will be postponed than the goal gene amplification curve, or interior be normal with reference to the result for Yintu(K19); Yet goal gene and interior when all being cloudy with reference to the gene result, it is invalid that this experiment is considered as, and needs to repeat again.

Positive control and negative control principle:

Each experiment all need be done positive control simultaneously.The positive control result is sun, shows that the detection system to target gene is normal; And when the result was the moon, it was invalid to look this time experiment, needed to repeat.

For whether proof has to pollute exist, each experiment also need be done negative control simultaneously.The negative control result is cloudy, shows that this experiment is pollution-free; Be sun as the result, it is invalid then to look this time experiment, needs to repeat.

1, primer and probe:

The HBV genom sequence of transferring in the Genebank database is compared, obtain target DNA fragment with Primerpremier5.0 and Oligo6.0 design PCR primer amplification, design specific probe in this fragment simultaneously.Require each probe Tm value to differ and be no more than 5 ℃, under same temperature, have optimum hybridization sensitivity and specificity.

Probe 5 ' end and 3 ' end are fluorescein-labelled, and primer and probe are the synthetic oligonucleotide, and be synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.The synthetic back that finishes of sequence is checked by the associate, is diluted to desired concn then as required.

2, Taq archaeal dna polymerase, UNG enzyme, dN (U) TP:

The Taq archaeal dna polymerase uses company of Takara company product.

DN (U) TP uses Shanghai Pu Luomaige (Promega) Bioisystech Co., Ltd product, and it is standby to be diluted to the working concentration packing.

The UNG enzyme uses Shanghai Pu Luomaige (Promega) Bioisystech Co., Ltd product.

3, the composition of work reference material:

Take the clinical serum of deactivation from chain hospital, with the hbv nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) that reaches peace gene limited-liability company that generally uses clinically, reagent lot number: 2010001, with the feminine gender of hepatitis B virus (HBV) the nucleic acid quantification standard substance (lot number 0711) of Nat'l Pharmaceutical ﹠ Biological Products Control Institute and positive serum as reference, the serum of bringing back from chain hospital is detected again, and calibrating to concentration is that 1 * 108IU/mL is standby.

4, positive reference material:

(original concentration is 1 * 108IU/mL) to be diluted to 1 * 107IU/mL and to be the strong positive quality control product with the clinical serum of the positive.

(original concentration is 1 * 108IU/mL) to be diluted to 1 * 104IU/mL and to be weak positive quality control product with the clinical serum of the positive.

5, positive quantitatively reference material:

The positive quantitatively reference material of HBV is to adopt the positive clinical serum of HBV, and warp quantitatively serum standard panel (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) is demarcated and obtained.Scaling method is the real-time quantitative fluorescence PCR method.

Positive quantitatively reference material DNA concentration is respectively 1 * 107IU/mL, 1 * 106IU/mL, 1 * 105IU/mL, 1 * 104IU/mL.

6, negative reference material

Be contrast with national reference material, adopt commercially available fluorescent quantitation product to detect negative clinical serum sample, at least 8 kinds.

N1-N8: feminine gender.

Use these 8 kinds of negative reference materials to detect, it is negative entirely that all 8 parts negative reference material detected results must be permitted, and false positive must not occur.

7, precision reference material

Be made up of the HBV serum sample, the final concentration of HBV serum sample is 1 * 104IU/mL, 1 * 106IU/mL.Use the precision reference material to detect, repeat 10 times, quantitatively the variation coefficient CV (%) of Ct value is respectively less than 15%; Worst error is not more than 300%.

8, sensitivity reference material

System adopts the HBV positive serum, and (1 * 108IU/ml), warp quantitatively serum standard panel (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) is demarcated and is obtained, and scaling method is the real-time quantitative fluorescence PCR method.

For the contrast fluorescent quantitation detects negative serum above-mentioned serum sample dilution is concentration shown in the linear sensitivity reference material of middle inspection (L0-L6) in order to national reference material.

Reference material is formed as table 1:

Table 1

Two, PCR reaction solution production technique

Formed i.e. dosing preparation, dosing and packing by three parts through determining this test kit PCR reaction solution production technique.Determine that according to the reaction system result of study this test kit PCR reaction solution prescription is as table 2:

Table 2

Reagent	Concentration	Volume (ul)
			Ultrapure water	-	31.5
PCR buffer	10×	5
			MgCl2	25mM	6
dATP	100mM	0.1
			dGTP	100mM	0.1
dCTP	100mM	0.1
			dUTP	100mM	0.2
D1	100uM	0.2
			D2	100uM	0.2
D3	100uM	0.2
			D4	100uM	0.2
D5	100uM	0.1
			D6	100uM	0.1
Total amount		44

Get a liquid mixing bottle, multiply by preparation people umber by the amount of the single every kind of reagent of dosing, mix.Above-mentioned PCR reaction solution is sub-packed in the 1.5mL centrifuge tube by every pipe 1056 μ L respectively, performs sign, preserve in the refrigerator below-18 ℃.

HBV DNA extraction liquid is produced

The used HBV DNA extraction liquid of the present invention comprises through determining this test kit HBV DNA extraction liquid production technique is made of i.e. dosing preparation, dosing and packing three parts.

Determine that according to the reaction system result of study this test kit DNA extraction liquid comprises extracting solution A and extracting solution B.HBV DNA extraction liquid A fills a prescription as table 3:

Table 3

The component composition	Every 1000ml
		PEG6000	100g
NaCl	25g
		The sterilization purified water	Be settled to 1000ml

DNA extraction liquid B prescription is as table 4.

Table 4:

The component composition	Every 1000ml
		NaOH	1g
1mol/L Tris-HCl pH 8.3	10ml
		NP40	10ml
Triton-100	10ml
		Chelex100	20g
0.5mol/L EDTA pH 8.0	0.2ml
		The sterilization purified water	Be settled to 1000ml

DNA extraction liquid A is sub-packed in the 1.5mL centrifuge tube with the 1mL/ pipe, and DNA extraction liquid B is sub-packed in the 1.5mL centrifuge tube with the 1mL/ pipe, 2-8 ℃ of storage.Test kit is formed (component of different lot numbers can be exchanged use in the test kit) as shown in table 5.

Table 5

Embodiment 2, hepatitis B virus PCR detection method of the present invention

One, reagent is prepared (reagent area in preparation)

(1) take out DNA extraction liquid B, standby.

(2) after definite reaction tubes that need carry out is counted n (number of samples+feminine gender+strong positive quality control product+weak positive quality control product+4 quantitative reference material), take out the HBV-PCR reaction solution, with n * 44 μ lHBV-PCR reaction solutions, n * 1 μ l Taq DNA Polymerase adds in the centrifuge tube and shakes mixing, instantaneous centrifugal after, packing 45 μ l in each PCR reaction tubes transfer to sample application zone behind the lid upper tube cap, and it is standby that lucifuge is put 4 ℃ of refrigerators.Reference substance and quantitative reference material are transferred to the sample preparation district, be put in 4 ℃ of refrigerators standby.

Two, DNA extraction (sample process district)

1, sample disposal:

1.1 get the DNA extraction liquid A that 100 μ l serum add equivalent, vibrator vibration mixing 5 seconds.

1.2 centrifugal 10 minutes of 10,000rpm.

1.3 remove supernatant, precipitate not obviously, need to suct clearly at centrifuge tube bottom settlings offside, should disposable exhaustion supernatant, avoid stirring or running into precipitation.

1.4 add 30 μ l DNA extraction liquid A (extracting solution includes water-fast material, fully draws behind the mixing) in the precipitation, vibrator thermal agitation mixing 5-10 second, instantaneous centrifugal several seconds, 100 ℃ of constant temperature processing 10 ± 1 minutes.

1.510, centrifugal 5 minutes of 000rpm, standby.

2, negative quality control product is handled:

2.1 take out negative quality control product, the centrifugal several seconds of 10,000rpm, inhale 50 μ l to the centrifuge tube of sterilizing, add 50 μ l DNA extraction liquid A, vibrator vibration mixing 5 seconds.

2.2 all the other steps are seen 1.2-1.4.

2.3HBV weak positive quality control product is handled (with negative quality control product).

2.4HBV the strong positive quality control product is handled (with negative quality control product).

2.5HBV positive quantitatively reference product is handled: the vibration mixing, the centrifugal several seconds of 10,000rpm, standby.

Three, pcr amplification

(1) application of sample (sample preparation district or sample application zone)

Add sample (comprising sample, negative quality control product, weak positive quality control product and strong positive quality control product) the supernatant liquor 5 μ l after handling in the ready PCR reaction solution pipe respectively, or directly add positive quantitative reference material 5 μ l, instantaneous centrifugal standby.

(2) pcr amplification (detection zone)

Ready PCR reaction tubes is put into the instrument sample cell, edit sample information and carry out amplified reaction by table 6 loop parameter.

Table 6

Reference value (term of reference)

Utilize the instrument software kit to analyze automatically, obtain each sample Ct value and C value (quantitatively reference value) as table 7.

Table 7

Four, interpretation of result condition enactment:

1.ABI 7500 baselines (baseline) are set: get 2 and arrive preceding 3 cycle values of sample threshold value (threshold) as baseline.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative control product amplification curve (random noise line), and namely Ct negative control=40 or " Undet. " are as the criterion.

2.STRATAGENE the Mx3000P baseline is set: select " being fit to baseline (Adaptive baseline) " fluorescent signal when setting.Threshold value (threshold) setting principle is with the vertex of threshold line just above normal negative control product amplification curve (random noise line), and namely Ct negative control=40 or " No Ct " are as the criterion.

Quality control standard:

This test kit positive and negative meet the following conditions to correlating simultaneously, otherwise it is invalid to be considered as this experiment:

1. negative Quality Control: HBV-FAM Ct value=40 or " No Ct " (Mx3000P) or " Undet. " (ABI 7500), interior reference (HEX) Ct value＜40, and logarithmic growth curve is preferably arranged.

2. positive quality control: HBV-FAM Ct value＜40, logarithmic growth curve is preferably arranged, and the strong positive quality control product quantitatively with reference to after between 1.00E+006-2.00E+007 (IU/mL); The weak quantitative reference value of positive quality control product between 2.00E+002-2.00E+004 (IU/mL), interior reference (HEX) Ct value≤40 (1.00e+006 is 1.00 and multiply by 10 6 powers).

3. positive quantitatively reference product: total positives, and linearly dependent coefficient 0.97≤| r|≤1;

Need more than to require in once testing, satisfying simultaneously, otherwise this experiment is invalid, need carry out again.

The result judges:

Utilize the instrument software kit to analyze automatically, obtain each sample Ct value

1. if the not S-type curve of HBV-FAM growth curve or Ct value=40 or " No Ct " are (Mx3000P) or " Undet. " (ABI 7500), in with reference to (HEX) channel C t value＜40, and have preferably the logarithmic growth curve then declare the HBV DNA total content of sample less than the detection lower limit.

2. if S-type and Ct value＜40 of HBV-FAM growth curve, then judgement by the following method:

If quantitative value is in linearity range: 5.00E+02≤C≤1.00E+08, then sample HBV DNA total content is C IU/mL.

If quantitative values is beyond linearity range, two kinds of situations are arranged: 1) C＞1.00E+08, sample HBV DNA total content＞1.00E+008IU/mL then is if the accurate quantification result can detect the diluted sample after extracting to linearity range again.2) C＜5.00E+02, then the HBV DNA total content of sample is C IU/mL, quantitative data is only for reference.

3.HBV-FAM:Ct value=40 or " No Ct ", interior reference (HEX) Ct value=40 or " NoCt ", the amplification failure please redeterminate.

See also Figure of description by this product accuracy, specificity, repeatability, stability, sensitivity and precision are studied, show that it possesses following performance:

The positive reference material detected result of Fig. 1 figure: 9 parts of positive reference material P1-P9 detection coincidence rates are 100% in the national standard product.

The interior reference of the positive reference material of Fig. 2, interior is sun with reference to the result, PCR reaction system and operation are normal.

Fig. 3 is specificity reference material detected result figure: 8 parts of specificity reference material N1-N8 detection coincidence rates are 100% in the national standard product.

The interior reference of the positive reference material of Fig. 4, interior is sun with reference to the result, PCR reaction system and operation are normal.

Fig. 5 is sensitivity reference material detected result figure (interior is sun with reference to the result, and PCR reaction system and operation are normal): 7 parts of linearities and sensitivity reference material meet following table 8 in the national standard product:

Table 8

Numbering	Quality standard (IU/mL)	The theoretical concentration logarithmic value
			L0	7.762E+07～6.465E+08	8.342
L1	1.479E+07～1.175E+08	7.619
			L2	1.585E+06～1.259E+07	6.649
L3	1.659E+05～1.318E+06	5.672
			L4	1.820E+04～1.479E+05	4.715
L5	1.514E+03～1.230E+04	3.636
			L6	3.890E+01～3.090E+02	2.043

Fig. 6: typical curve 1e4-1e7 reference product detected result figure.

Fig. 7 is the typical curve made from Fig. 6 result.

Fig. 8: precision detected result figure (repeat 10 times, interior is sun with reference to the result, and PCR reaction system and operation are normal).

Fig. 9: be strong positive and weak positive quality control product detected result figure (interior is sun with reference to the result, and PCR reaction system and operation are normal).

To sum up, compare with laboratory regular-PCR detection by quantitative, this product accuracy height, high specificity, and in repeatability, stability, sensitivity and precision very big improvement has been arranged, and improving, keeping life is 6 months, product performance are stable before the deadline.

Embodiment 3, clinical trial analysis of the present invention:

For this product is detected, choose Fuzhou City Infectious Disease Hospital, Fujian Provincial Hospital and three three grades of first-class clinical hospitals of family of 174 hospitals of PLA 08 month in October, 2010 in 2010 and verify for clinical verification unit.Concrete examination arrangement sees Table 9:

Table 9

The result:

The test of three tame hospitals detects 649 parts of serum specimens altogether, wherein use control test reagent, the hbv nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) that is Da An genome company detects, positive sample has 343 parts, negative sample has 306 parts, recall rate is 53%, specifically sees Table 10, table 11, table 12.

Table 10

	Safe general detected result	Control test result
			Positive routine number	344	343
Negative routine number	305	306
			Add up to routine number	649	649
Positive rate	53％	52.85％

Table 11

The interval distribution	Tai Pu result's (part)	Results of comparison (part)
			5.0×10 ²～9.9×10 ²IU/mL	23	24
1.0×10 ³～9.9×10 ³IU/mL	78	82
			1.0×10 ⁴～9.9×10 ⁴IU/mL	56	54
1.0×10 ⁵～9.9×10 ⁵IU/mL	48	46
			1.0×10 ⁶～9.9×10 ⁶IU/mL	48	52
1.0×10 ⁷～9.9×10 ⁷IU/mL	86	77
			＞1.0×10 ⁸IU/mL	5	8
Add up to	344	343

Table 12

By table 10, table 11, table 12 data as calculated, be subjected to trial product clinical study result as follows:

Sensitivity=99.42%;

Specificity=99.02%;

Coincidence rate (crude agreement)=99.23%;

Positive findings predictive value=99.13%;

Negative findings predictive value=99.34%;

False positive rate (misdiagnosis rate)=0.98%;

False negative rate (rate of missed diagnosis)=0.58%;

Youden index (correct index)=0.9844;

Recall rate=53.00%;

Can draw thus, the positive coincidence rate of safe general reagent and contrast agents detected result is 99.42%, with the negative match-rate of contrast agents detected result be 99.02%, total coincidence rate is 99.23%.The general hbv nucleic acid amplification detection by quantitative test kit of Thailand (fluorescent PCR method) mainly is present on some very low indivedual samples that copy with the inconsistent of detected result of the hbv nucleic acid detection by quantitative test kit (PCR-fluorescent probe method) that reaches the peace gene, and these samples are referred again to the detected result that hospital is born in clinical trial.

Below bear the Clinical Laboratory numbering of hospital according to clinical experiment, provide the detection case of the discrepant sample of these detected results, shown in table 13 (detected result of provincial hospital, Fujian Province difference sample), table 14 (detected result of Fuzhou City, Fujian Province infectious hospital difference sample), table 15 (detected result of No.174 Hospital, PLA's difference sample).

Table 13

Table 14

The sample numbering	Safe general detected result	Reach safety check and survey the result	Hospital's detected result
				0531134	N	6.84E+02	＜4.2E+2
0531166	3.55E+02	1.42E+03	＜4.2E+2

Table 15

The sample numbering	Safe general detected result	Reach safety check and survey the result	Hospital's detected result
				520282301	1.88E+02	5.11E+02	1.34E+02
520279446	6.77E+02	4.80E+02	6.85E+02

From table 13, table 14, table 15 as can be seen, in 649 parts of serum specimens, safe general reagent with reach peace reagent detected result have 7 parts inconsistent, be the low-down weak positive of negative sample or copy number.Safe general reagent detects negative, and hospital's detected result is also negative; Safe general test positive, hospital's detected result is also positive, and safe general detected result and hospital's detected result fit like a glove.

In process of the test, use same group of sample of the parallel detection with contrast agents of safe general reagent respectively, carry out paired t-test again, t=0.726, P=0.468＞0.05 illustrates that both quantitative results do not have significant difference; Simultaneously, the result with two kinds of reagent is the X/Y axle respectively, sets up straight-line equation and calculates correlation coefficient r, and r=0.9805 illustrates that both results are linearly relevant.In addition, as quantitative Diagnosis reagent, we also divide negative, positive according to the Cut off value of amplification, and two groups of results are matched χ2Jian Yan.The pairing χ2Jian Yan, χ 2=0, the a=0.05 level press in P＞0.05, and the difference not statistically significant can be thought the detected result indifference of two kinds of detection methods, safe general reagent and the control test reagent equivalence of having gone on the market.

The hbv nucleic acid detection by quantitative test kit (fluorescent PCR method) of verifying the development of Tai Pu company by synchronous blind trial has its described functional characteristics, and the result accurately and reliably, and is easy to operate, is applicable to the clinic diagnosis of chronic HBV infection.

This test kit has higher specificity and sensitivity, can directly monitor the situation that copies of hepatitis B virus nucleic acid in the serum (HBV-DNA), can difference diagnose the early infection of hepatitis B, existing disease infection, decubation to infect, also can diagnose the mutant strain of hepatitis B, and active the copying of virus is the factor that starts or excite the liver organization inflammatory reaction.

The above only is embodiments of the invention; be not so limit claim of the present invention; every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.

Claims

1. a hepatitis B virus PCR fluorescence quantitative detection kit comprises the PCR reaction solution, it is characterized in that, described PCR reaction solution contains primer A and fluorescent probe A; Described primer is divided into upstream primer A and downstream primer A:

Upstream primer A:5 '-TCTTCTTGTTGGTTCTTCTGGACT-3 ';

Downstream primer A:5 '-AAACATAGAGGTTCCTTGAGCAGG-3 ';

Fluorescent probe A:5 '-CAAGGTATGTTGCCCGTTTGTCCTCTA-3 ';

Described test kit also comprises DNA extraction liquid, archaeal dna polymerase, UNG enzyme, strong positive quality control product, weak positive quality control product, negative quality control product, positive quantitatively reference product;

Described PCR reaction solution has been introduced an artificial constructed interior reference system and has been used for avoiding the detected result false negative, reference system is reference system in the Arabidopis thaliana in described, described in reference system comprise upstream primer B sequence as: SEQ ID NO.4, downstream primer B sequence as: SEQ ID NO.5 and fluorescent probe B sequence are as SEQ ID NO.6.