CN102827949A - Measles virus RT-tHDA detection kit and detection method - Google Patents

Abstract

The invention provides a measles virus nucleic acid helicase-dependent isothermal amplification detection kit and a detection method. The kit mainly comprises an RT-tHDA reaction reagent and a specific amplification primer; the specific amplification primer comprises: an upstream primer, 5'-AACACGAACAGATGACAAGTTGCGAAT-3'; and a downstream primer, 5'-TGTTATCCTTCAATGGTGCCCACTC-3'. The method of the invention has high specificity on measles virus detection, has no cross reaction with other respiratory viruses, has high sensitivity, can detect measles virus nucleic acid directly from samples of mouthwash, throat swabs and urine of suspected measles patients, only needs about 3 hours from virus nucleic acid extraction to detection completion, and is quite suitable for laboratory early stage diagnosis of sudden epidemic situation caused by measles virus infection.

Description

A kind of Measles virus RT-tHDA detection kit and detection method

(1) technical field

The present invention relates to a kind of Measles virus RT-tHDA detection kit and detection method.

(2) background technology

Measles be by Measles virus cause a kind of be the acute infectious disease of characteristic with heating, respiratory symptom and eruption.Since the seventies in 20th century, the whole world implemented to enlarge The Immune Programming (EPI), whole world measles sickness rate, mortality ratio descended significantly, but many Sporadic cases still occur every year at present, and usually cause partial popular.According to the job requirement of China's elimination measles, the measles case that occurs must be confirmed the chamber of experimentizing.

(3) summary of the invention

The object of the invention provides a kind of test kit and detection method that relies on helicase isothermal amplification technology rapid detection Measles virus nucleic acid.

The technical scheme that the present invention adopts is:

A kind of rt of Measles virus nucleic acid relies on heat-resisting helicase constant-temperature amplification (RT-Thermophilic Helicase-dependent Amplification; RT-tHDA) test kit; Mainly comprise mainly comprising RT-tHDA reaction reagent and specificity amplification primer, said specificity amplification primer is following:

Upstream primer: 5 '-AACACGAACAGATGACAAGTTGCGAAT-3 ';

Downstream primer: 5 '-TGTTATCCTTCAATGGTGCCCACTC-3 '.

The key of test kit of the present invention is selection of primers, and the RT-tHDA reaction reagent is a used conventional reagent in the RT-tHDA reaction, like IsoAmp Enzyme Mix, MgCl2, hermoScript ^TMReverse Transcriptase etc., these components belong to common practise to those skilled in the art, can prepare voluntarily as required, and HAD test kit when perhaps directly buying is like HDA test kit of Biohelix company etc.

The invention still further relates to and utilize said test kit to detect the method for Measles virus, said method comprises:

(1) extracts testing sample RNA; Said sample RNA extracts and can be undertaken by ordinary method, as adopts RNeasy Mini Kit or other test kit of German QIAGEN company, extracts according to the test kit specification sheets.

(2) be template with testing sample RNA, add specificity amplification primer and PCR reaction reagent, be made into the RT-tHDA reaction system, carry out amplified reaction;

(3) the RT-tHDA reaction product is carried out agarose gel electrophoresis and detect,, then contain Measles virus in the testing sample if come across the electrophoretic band of 110bp size; Otherwise then deny.

Among the present invention, said amplification reaction condition is: 65 ℃, and 120min.

Said RT-tHDA reaction system final concentration is formed as follows:

The heat-resisting reversed transcriptive enzyme ThermoScript of Invitrogen company ^TMReverse Transcriptase0.5 μ l

Add water polishing to 50 μ l.

At present; The domestic method of measles is confirmed in the laboratory; Comprise that serology detects and the etiology detection, the former is with enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, ELISA) the specificity measle immune globulin (Immunoglobulin in the detection patient serum; Ig) M, the latter confirm with detection Measles virus nucleic acid to separate Measles virus.General virus separation, reverse-transcription polymerase chain reaction (the Reverse Transcription-Polymerase Chain Reaction of adopting; RT-PCR) or fluorescent quantitative RT-PCR method carry out detection of nucleic acids; But, when being promoted in basic unit, it receives certain restriction because instrument cost is higher.The nucleic acid isothermal amplification technique of the dependence helicase that developed recently gets up [Helicase-dependent Isothermal Deoxyribonucleic Acid (DNA) Amplification; HAD]; The natural process of dna replication dna is all carried out the whole process of its amplified reaction in the analogue body under single temperature, need not special amplification instrument; Overcome the working cycle of tens temperature variation of PCR reaction needs experience, used in being particularly suitable for detecting at the scene.The present invention is design specific primers voluntarily; The rt of having set up rapid detection Measles virus nucleic acid relies on heat-resisting helicase constant-temperature amplification (RT-Thermophilic Helicase-dependent Amplification; RT-tHDA) method is stated the result as follows at present.

The application contriver has downloaded the Measles virus nucleotide sequence that recent two decades comes all over the world from the NCBI gene pool of the U.S.; It has been carried out homology relatively; Hemagglutinin gene design at Measles virus nucleic acid is some to primer, and specific amplification is carried out in this zone, therefrom filters out best primer; And the RT-tHDA method is optimized, verify its susceptibility, specificity and repeatability.Through respectively the detection of mumps virus, rubella virus, respiratory syncytial virus and suspected patient clinical sample being compared; This method has high specific; Can only detect Measles virus nucleic acid, with the equal no cross reaction of other Respiroviruses, and also quicker and easy than conventional RT-PCR method.With the novel method of setting up, to recent Zhejiang Province 21 parts of clinical samples of doubtful measles patient chamber quick diagnosis that experimentizes, obtained gratifying result, in time take measure of control for this disease and brought into play good effect.

Beneficial effect of the present invention is mainly reflected in: the inventive method has specificity highly to the detection of Measles virus, with viral no cross reactions such as other Respirovirus mumps viruses, rubella virus, respiratory syncytial virus; It is highly sensitive that the inventive method detects, and can be directly from samples such as doubtful measles patient's GARG, throat swab, urine, detects Measles virus nucleic acid, is extracted into from viral nucleic acid and accomplishes detection and only need about 3h.

(4) description of drawings

Fig. 1 .RT-tHDA method detects Measles virus specificity result; M: Φ X174-Hinc II DNAMarker; 1: Measles Vaccine strain Shanghai 191; 2: measles epidemic strain Zhejiang/05/08; 3: rubella virus; 4: mumps virus 5.EV71; 6: respiratory syncytial virus.

Fig. 2 is different extent of dilution Measles virus results for the RT-tHDA method detects;

Fig. 3 is different extent of dilution Measles viruss for the RT-PCR method detects;

Among Fig. 2, Fig. 3:

M: Φ X174-Hinc II DNA Marker; 1 virus concentration 5.012 * 10 ^-7Mol/L; 2 virus concentration 5.012 * 10 ^-8Mol/L; 3 virus concentration 5.012 * 10 ^-9Mol/L; 4 virus concentration 5.012 * 10 ^-10Mol/L; 5 virus concentration 5.012 * 10 ^-11Mol/L; 6 virus concentration 5.012 * 10 ^-12Mol/L.

Fig. 4 is the result who directly from clinical samples, detects Measles virus;

M. Φ X174-Hinc II DNA Marker; 1: negative control; 2: Measles Vaccine strain Shanghai 191; 3～6 fluorescence quantitative RT-RCRs detect positive sample; 7: the fluorescence quantitative RT-RCR negatives.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

Materials and methods

1.1 experiment virus strain Measles Vaccine strain Shanghai 191 (Shanghai, Shanghai; S191), the viral Jerry Lynn of mumps (parotitis) (JL) strain, rubella virus GOS strain, respiratory syncytial virus Long strain is provided by Shanghai Vaccine and Serum Institute and National Institute for Food and Drugs Control respectively.Measles virus epidemic strain Zhejiang (Zhejiang; ZJ)/05/08, enterovirns type 71 (Enterovirus Type71, EV71) strain; And doubtful measles patient's GARG, throat swab, urine etc., provide by virus institute of Zhejiang Center For Disease Control and Prevention (CDC).

1.2 design of primers is compared to the Measles virus different genotype, chooses the common conservative region design primer of each genotype virus hemagglutinin gene, to guarantee the versatility of primer.Influenced by the dna helicase ability of unwinding, RT-tHDA product size be controlled at 80 base pairs (Base Pair, bp)～120bp; Primer length is at 24bp～30bp, and (Melting temperature Tm) is arranged on 62 ℃～74 ℃ to the DNA melting temperature (Tm); Cytosine(Cyt) and guanine (Guanine and Cytosine; GC) per-cent is avoided secondary structure 40%～60% in the primer, does not have complementary sequence between primer with in the primer; And (Basic Local Alignment Search Tool BLAST) carries out specificity to institute's designed primer and confirms to use research tool based on local alignment algorithm.

Design a pair of Auele Specific Primer according to above condition:

F:5’-AACACGAACAGATGACAAGTTGCGAAT-3’;

R:5’-TGTTATCCTTCAATGGTGCCCACTC-3’。

The amplified fragments size is 110bp, and primer is by Nanjing Genscript Biotechnology Co., Ltd.'s synthesizing and purifying.

1.3 extraction Measles virus Yeast Nucleic Acid (the Ribonucleic Acid of viral nucleic acid; RNA) nucleic acid extraction kit (High Pure Viral Nucleic Acid Kit) of German Roche company is adopted in extraction; Extract viral RNA according to the test kit specification sheets, place-70 ℃ of preservations subsequent use.

1.4RT-tHDA reaction conditions RT-tHDA adopt the Biohelix HDA of company test kit and add heat-resisting reversed transcriptive enzyme and react, reaction system 50 microlitres (μ l), wherein 10 * Annealingbuffer II5 μ l, MgSO ₄Each 3.5 μ l of [100 mmoles (mM)] 1.75 μ l, NaCl (500mM) 4 μ l, IsoAmpdNTP Solution and IsoAmp Enzyme Mix, sample RNA2 μ l, each 0.75 μ l of 20 μ M upstream and downstream primers, the heat-resisting reversed transcriptive enzyme ThermoScript of Invitrogen company ^TMReverse Transcriptase0.5 μ l adds ddw to 50 μ l.Behind the reaction solution mixing, place 65 ℃ of 120min to react after, get 5 μ l with 0.02 grams per milliliter (g/ml) agarose gel electrophoresis observations.

1.5 conventional RT-PCR reaction:

Measles virus nucleotide sequence primer

F:5’-AACACGAACAGATGACAAGTTGCGAAT-3’;

R:5’-TGTTATCCTTCAATGGTGCCCACTC-3’

Amplification segment 110bp.Adopt the TAKARA one step RT-kit of company (code:DRR024A), press the test kit specification sheets and operate, reaction system is 25 μ l, 10 * RT-PCR damping fluid 2.5ul wherein, MgCl ₂2.5ul (25mM), dNTP mixture (each 10mM) 2.5 μ l, RNase suppressor factor (40U/ μ l) 0.5 μ l; AMV enzyme (5U/ μ l) 0.5ul, Taq enzyme (5U/ μ l) 0.5ul, each 0.5 μ l of the upper reaches and downstream primer (20 μ M); Template ribonucleic acid 2 μ l, DEPC water 4.5 μ l.Reaction conditions is 50 ℃ of 30min, and 95 ℃ of 3min carry out rt, 95 ℃ of 20s then, and 50 ℃ of 25s, 72 ℃ of 30s increase, and change 72 ℃ of 10min after 40 circulations over to, get to judge behind the 5 μ l product electrophoresis and have or not specific band (110bp).

1.6RT-tHDA specificity, susceptibility and replica test

Respectively Measles Vaccine strain Shanghai 191, measles epidemic strain Zhejiang/05/08, mumps virus, rubella virus, EV71, respiratory syncytial virus are extracted nucleic acid, the RT-tHDA method of setting up with the present invention detects, relatively the specificity of its detection.With NanoVue spectrophotometric instrumentation Measles virus nucleic acid original liquid concentration, nucleic acid stoste is carried out 10 times of gradient dilutions to 10 ^-5, adopt same primers as and reaction volume, parallel RT-tHDA and RT-PCR reaction, the relatively susceptibility of two kinds of nucleic acid amplification methods of carrying out.

2 results

2.1 specificity test

The result sees Fig. 1, and is visible, and the RT-tHDA method that the present invention sets up has specificity preferably to Measles virus, to no cross reactions such as other Respiroviruses such as mumps virus, rubella virus, respiratory syncytial virus.

2.2 sensitivity test

With NanoVue spectrophotometric instrumentation Measles virus nucleic acid original liquid concentration, nucleic acid stoste is carried out 10 times of gradient dilutions to 10 ^-5, adopt same primers as and reaction volume, parallelly carry out RT-tHDA and RT-PCR reaction, the susceptibility of two kinds of nucleic acid amplification methods of comparison, the result sees Fig. 2, Fig. 3.

The result is visible, and the RT-tHDA method that the present invention sets up is worked as susceptibility and the RT-PCR reacting phase of Measles virus, and minimum detectable concentration is 10-10mol/L.

2.4 the detection of clinical sample

GARG from various places, recent Zhejiang Province measles suspected patient; Throat swab and urine are directly extracted viral RNA in totally 21 parts of clinical samples; Detect simultaneously with RT-tHDA of the present invention and fluorescent quantitative RT-PCR method (seeing operation 1.5); RT-tHDA method of the present invention as a result detects positive 20 parts of Measles virus nucleic acid, and the fluorescence quantitative RT-RCR method detects positive 20 parts of measles nucleic acid, and the positive rate that Measles virus RT-tHDA method of the present invention and fluorescence RT-PCR method detect Measles virus is suitable.Measles virus RT-tHDA method of the present invention is used for the checking of clinical sample detection carries out 4 parallel laboratory test chambers, has all obtained satisfied result, and what Fig. 4 showed is the detection collection of illustrative plates of clinical sample.

Claims (3)

1. a Measles virus RT-tHDA detection kit mainly comprises RT-tHDA reaction reagent and specificity amplification primer, it is characterized in that said specificity amplification primer is following:

Upstream primer: 5 '-AACACGAACAGATGACAAGTTGCGAAT-3 ';

Downstream primer: 5 '-TGTTATCCTTCAATGGTGCCCACTC-3 '.

2. utilize the said test kit of claim 1 to detect the method for Measles virus, said method comprises:

(1) extracts testing sample RNA;

(2) be template with testing sample RNA, add specificity amplification primer and PCR reaction reagent, be made into the RT-tHDA reaction system, carry out amplified reaction;

(3) the RT-tHDA reaction product is carried out agarose gel electrophoresis and detect,, then contain Measles virus in the testing sample if come across the electrophoretic band of 110bp size; Otherwise then deny.

3. method as claimed in claim 2 is characterized in that said amplification reaction condition is: 65 ℃, and 120min.

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