CN101597653A - Fluorescence quantitative PCR detection kit for hepatitis B virus - Google Patents

Abstract

The present invention relates to the hepatitis B virus detection range, a kind of fluorescence quantitative PCR detection kit for hepatitis B virus is disclosed, this test kit comprises: upstream outer primer, downstream outer primer, upstream inner primer, downstream inner primer and the Taqman probe of the conservative section of specific amplification hepatitis B virus.Utilize test kit of the present invention that hepatitis B virus is detected, have highly sensitive, high specificity, advantage such as fast and convenient, can be by treating the absolute quantitation of this HBV of sample copy number, for clinical diagnosis and treatment provide important reference.

Description

Fluorescence quantitative PCR detection kit for hepatitis B virus

Technical field

The present invention relates to the hepatitis B virus detection range, relate in particular to a kind of fluorescence quantitative PCR detection kit for hepatitis B virus.

Background technology

Hepatitis B virus (hepatitis B vrius HBV) infects and to be worldwide popular, be Type B viral hepatitis main diseases because of.China is high incidence of hepatitis b, and 9.75% population i.e. 1.3 hundred million people is hepatitis B surface antigen (HBsAg) carrier, and hepatitis is added up and surpassed 4,000 ten thousand.Because the highly infective of HBV, the detection of HBV are clinical and the emphasis of blood safety control always.

Clinically to the detection of HBV based on serology and gene test.Wherein PCR method is adopted in gene test more, and the most basic factor of restriction PCR reaction sensitivity is the quantity of pcr amplification reaction template, if quantity is very little, and PCR reaction failure usually.To this, the template number that people adopt the method for two steps nest-type PRC amplification artificially to increase pcr amplification reaction usually improves sensitivity.Two step nest-type PRCs are made up of a pair of outer primer and a pair of inner primer at two pairs of amplimers of a target fragment design, and the external primer amplification fragment comprises the inner primer amplified fragments.Working method is carried out pcr amplification for using earlier a pair of outer primer, is that the template of PCR is carried out the secondary amplification with inner primer with this an amount of pcr amplification product then, thereby is called " nest-type PRC " visually.This method can make sensitivity improve 1 order of magnitude usually.But this method will be carried out 2 operations, comparatively trouble to same sample.And because the template of the amplification second time is the product that increases for the first time, the preceding PCR reaction tubes of secondary amplification is uncapped and handled is the main source of laboratory pollution, credibility for the result has serious negative impact, thus be clinical detection and blood screening should avoid.The highest external product of its sensitivity of fluorescent quantitative PCR technique that is used for blood HBV detection at present is 300 copies/ml, and home products is 420 copies/ml.

Summary of the invention

For addressing the above problem, the object of the present invention is to provide a kind of fluorescence quantitative PCR detection kit for hepatitis B virus that can in same reaction tubes, realize the nido amplification, it is highly sensitive, stable strong that this test kit is used to detect hepatitis B virus.

For achieving the above object, the present invention adopts following technical scheme:

A kind of fluorescence quantitative PCR detection kit for hepatitis B virus, described test kit comprises:

1) the upstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:1;

2) the downstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:2;

3) the upstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:3;

4) the downstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:4;

5) be used to detect the Taqman probe that hepatitis B virus is guarded section, its base sequence is shown in SEQ ID NO:5.

Described Taqman probe 5 ' end flag F AM, 3 ' end mark TAMARA.

The present invention promptly realizes twice operation of two original PCR reaction tubess by to inside and outside two pairs of primer design " nest-type PRC " being achieved by the primary first-order equation in the PCR reaction tubes in same reaction tubes.This is designed to: the annealing temperature (T of outer primer _m) height, the annealing temperature (T of inner primer _m) low, enough low in the concentration of the about primer of PCR reaction solution, the concentration of inner primer is high relatively, simultaneously for the anti-pollution UNG enzyme that adds in the PCR reaction solution.

Because interior outer primer is to the difference of annealing temperature, outer primer works better in the time of 68 ℃ and inner primer can't be worked; And the amount of outer primer is enough low, reacts to remain little after 10 circulations, reduces annealing temperature to 60 ℃, suitable inner primer amplification again.Because outer primer has carried out 10 to the HBV target fragment and has taken turns amplification, these amplified production fragments unusual weak point for whole HBV genome, as inner primer PCR reaction template the time, has very strong advantage than HBV genome, and the theoretical quantity of external primer amplification product be protogene group quantity 2 10 powers doubly, be easy to cause amplified reaction with this template as the inner primer amplification.This method has been equivalent to increase the concentration of reaction template, thereby has improved the sensitivity that detects greatly.

Utilize the internal primer amplification fragment of fluorescent PCR technology indoor design Taqman hydrolysis probes.One step nest-type PRC is combined with Taqman fluorescence hydrolysis probes technology.Though also can be during external primer amplification to the Taqman probe hydrolysis, can produce fluorescent value, but amplification program only is designed to collect fluorescence in the extension stage of inner primer amplification, so the fluorescence that produces of external primer amplification is used as background and deducts, and whole amplification is had no effect.Moreover, by the amplification of 10 round-robin of outer primer in early stage, pcr amplification quite stable when entering the inner primer amplification, directly setting baseline when carrying out data analysis is 1-1, and need not to regulate the deviation when having reduced data analysis according to differences such as amplifing reagents.The present invention can realize the sensitivity of 100 copies/ml.

Utilize test kit of the present invention that hepatitis B virus is detected, have highly sensitive, high specificity, advantage such as fast and convenient, can be by treating the absolute quantitation of this HBV of sample copy number, for clinical diagnosis and treatment provide important reference.

Description of drawings

Fig. 1 is the national reference material fluorescence quantitative PCR detection figure as a result of 10 times of gradient dilutions;

Fig. 2 is diluted to 100Copies/mL serum sample fluorescence quantitative PCR detection figure as a result.

Embodiment

Embodiment 1

A kind of fluorescence quantitative PCR detection kit for hepatitis B virus, described test kit comprises:

1) the upstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:1;

2) the downstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:2;

3) the upstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:3;

4) the downstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:4;

5) be used to detect the Taqman probe that hepatitis B virus is guarded section, its base sequence is shown in SEQ ID NO:5, and Taqman probe 5 ' is held flag F AM, 3 ' end mark TAMARA.

Embodiment 2 usefulness embodiment 1 described test kit detects the national reference material of 10 times of gradient dilutions

The 40uL reaction system adds template DNA, the national reference material of 10 times of gradient dilutions (lot number: 0711) 2uL.

The final concentration of each composition is in the pcr amplification system: 1 * buffer (Promega)

3mMMg ²⁺

Each 200uM of dATP/dGTP/dCTP, dUTP400uM

0.1UUNG enzyme

2.5UTaq enzyme

Outer primer OF 0.1pmol

Outer primer OR 0.1pmol

Inner primer IF 10pmol

Inner primer IR 10pmol

Probe P 3pmol

The pcr amplification program is: 50 ℃ of 2mins;

95℃10mins；

95℃5secs，68℃35secs，10cycles；

95 ℃ of 5secs, 60 ℃ of 30secs, 30cycles reads glimmering

Light value.

Detected result is seen Fig. 1, L0=2.198 * 10 ⁸Copies/mL, L0/10, L0/10 ², L0/10 ³, L0/10 ⁴, L0/10 ⁵, L0/10 ⁶, R2=0.999888, good linearity.

Embodiment 2 usefulness embodiment 1 described test kit detects the serum sample that is diluted to 100copies/mL

The pcr amplification system is identical with pcr amplification condition and embodiment, and template is the serum sample that is diluted to 100copies/mL, repeats 12 times.

Detected result is seen Fig. 2, cv%=9.88%, and repeatability is high.

SEQUENCE?LISTING

＜110〉Yaneng Biotechnology (Shenzhen) Co., Ltd.

＜120〉fluorescence quantitative PCR detection kit for hepatitis B virus

<130>P10947

<160>5

<170>PatentIn?version?3.3

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＜213〉artificial sequence

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gaccaccaaa?tgcccctatc?ttatcaacac?t 31

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＜213〉artificial sequence

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ctcaatgtta?gtattccttg?gacacataag?gt 32

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＜213〉artificial sequence

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actactgttg?ttagacgaag?a 21

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＜213〉artificial sequence

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caggtcccct?agaagaagaa?ctcc 24

Claims (2)

1, a kind of fluorescence quantitative PCR detection kit for hepatitis B virus is characterized in that described test kit comprises:

1) the upstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:1;

2) the downstream outer primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:2;

3) the upstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:3;

4) the downstream inner primer of the conservative section of specific amplification hepatitis B virus, its base sequence is shown in SEQ ID NO:4;

5) be used to detect the Taqman probe that hepatitis B virus is guarded section, its base sequence is shown in SEQ ID NO:5.

2, fluorescence quantitative PCR detection kit for hepatitis B virus according to claim 1 is characterized in that: described Taqman probe 5 ' end flag F AM, 3 ' end mark TAMARA.

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