CN101935715A

CN101935715A - Method for detecting Chikungunya virus nucleic acid by real-time fluorescent quantitative PCR

Info

Publication number: CN101935715A
Application number: CN201010276645XA
Authority: CN
Inventors: 姚李四; 燕清丽; 张晓龙
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2010-09-08
Filing date: 2010-09-08
Publication date: 2011-01-05
Anticipated expiration: 2030-09-08
Also published as: CN101935715B

Abstract

The invention discloses a method for detecting Chikungunya virus nucleic acid by real-time fluorescent quantitative PCR, comprising: obtaining a primer and a probe, and detecting the Chikungunya virus nucleic acid. The invention has the advantages of good specificity, high sensitivity and the like.

Description

Real-time fluorescence quantitative PCR detects the method for datum hole Kenya viral nucleic acid

Technical field

The invention provides a kind of method of utilizing real-time fluorescence quantitative PCR to detect datum hole Kenya viral nucleic acid.

Background technology

Chikungunya virus (Chikungunya virus) is the sub-thread positive chain RNA virus, belongs to Togaviridae (Togaviridae) alphavirus (Alphavirus), cause chikungunya heat (Chikungunyafever, CHIK).This disease is a kind of acute infectious disease, propagates to the people by yellow-fever mosquito, and be feature with heating, arthralgia, fash and hyporrhea clinically.

Nineteen fifty-three is isolated Chikungunya virus first from 1 routine fever patient's of Tanzania blood.The 60-90 age in 20th century should disease all has popularly in many countries in Africa middle part and south, and is separated to CHIK virus, as the Sudan, Uganda, cd, Central African Republic, Malawi, Zimbabwe, Kenya and South Africa; The Senegal in West Africa, Benin, Republic of Guinea, Cote d'lvoire and Nigeria etc.; In South East Asia, Indonesia broke out CHIK first in 1999, and it is popular that this disease appears in 2001-2003 this place once more; On island, the Indian Ocean, in recent years, large-scale CHIK outbreak of epidemic appearred in island, the Indian Ocean.2005, big outbreak of epidemic and bamboo telegraph other island to the whole Indian Ocean took place in Comoros island; In Europe, in July, 2007, CHIK takes place in 2 villages that Italian Ravenna is economized; 2004-2007, geographic CHIK outbreak of epidemic such as Asia and Africa constantly take place, and wherein about 2,000,000 examples of CHIK patient take place in the whole world altogether in 2006.It is popular that Taiwan that it should be noted that China at 1967 and 2006 CHIK took place once, the CHIK introduced cases that China mainland in 2008 is found first.But China does not still have CHIK native country popular and definitely reports so far.Throughout history, CHIK has become the serious day by day public health problem in whole world district.

At present, what mainly adopt at the laboratory Fast Detection Technique of external transmissible disease is immunology detection and nucleic acid detection technique, is the nucleic acid detection technique of representative with the round pcr especially, and very sensitive detects the nucleic acid of various eqpidemic disease pathogenic agent.But because the pcr amplification technology is often brought the specificity problem, various countries scientist and technician on the round pcr basis, in conjunction with other new technology, make great efforts to improve the specificity and the susceptibility of round pcr.In recent years, the real-time fluorescence PCR technology that PCR-based technology and the technical development of ABI Taqman probe hybridization are got up has greatly been improved specificity and the susceptibility and the resistant to pollution ability of conventional P CR technology, in the inspection and quarantine that is applied to various eqpidemic diseases more and more widely.Along with further developing of probe technique is perfect, the appearance of a kind of novel Hydro Easy Probes can effectively improve the high shortcoming of Taqman probe background signal, and further improves the sensitivity that detects.The present invention intends adopting novel HydroEasy Probes designing technique, and the fluorescent PCR of exploitation Chikungunya virus detects diagnostic method, for the rapid detection of the Chikungunya virus of frontier port provides powerful guarantee.

Summary of the invention

The object of the present invention is to provide a kind of real-time fluorescence quantitative PCR to detect the method for datum hole Kenya viral nucleic acid.This method is utilized the real-time fluorescence quantitative PCR detection technique, has designed a pair of primer and probe Virus Sample is carried out the real-time quantitative detection.This method detection specificity is good, highly sensitive.

Real-time fluorescence quantitative PCR of the present invention detects the method for datum hole Kenya viral nucleic acid, at first comprise and obtain to detect the primer of datum hole Kenya viral nucleic acid and the step of probe, this step is specially: (1) collects screening datum hole Kenya virus whole genome sequence, utilize information biology software that it is carried out the homology comparison, at its conserved regions design primer; (2) design specific probe according to the nucleotide sequence in the pcr amplification zone, and login the specificity of detection probes among the GENEBANK; (3) thing to be detected is carried out real-time quantitative analysis.

In aforesaid method of the present invention, the datum hole Kenya virus of being screened can be all datum hole Kenya virus whole genome sequences that NCBI announces.In addition, in the method for the present invention, can utilize DNASTAR software to carry out the homology comparison of sequence.Comparison result is seen Fig. 1.Dash area is certain high conserved region in the complete sequence among the figure.

In primer of the present invention and probe design, at first the comparison sequence conserved regions design upstream and downstream primer, according to the design of primers principle, design upstream and downstream primer between conserved regions, wherein there is degeneracy in the 5th and the 25th at downstream primer, design is positioned at the special probe of amplification region, and wherein there is degeneracy in the 19th and the 21st at probe.Primer and probe are when synthetic, and probe 5 ' end connects the FAM fluorophor, and 3 ' end connects the non-fluorophor of BHQ.The specificity of primer strengthens.Primer and probe sequence see Table 1.

Table 1

Real-time fluorescence quantitative PCR of the present invention detects the method for datum hole Kenya viral nucleic acid, and next comprises the step that detects datum hole Kenya viral nucleic acid, and this step is specially:

(1) nucleic acid extraction

Viral RNA extracts selects for use QIAamp Mini kit 52906 viral RNAs to extract test kit.

(2) RT-PCR amplification

The amplification kit of selecting for use is the AgPath-ID of ABI company ^TMOne-step RT-PCR Kit.

Reaction system component and volume thereof see Table 2.

Table 2

Amplification condition: the standard amplification condition that adopts this area.

For example

45 ℃ 10 minutes

95 ℃ 10 minutes

In the method for the present invention, probe links to each other with fluorophor, and described fluorophor is FAM-BHQ, and other fluorophor is suitable for too, such as, FAM-TAMARA etc.

Described RT-PCR reacts spendable instrument and comprises ABI real-time PCR system (for example 7000,7300,7500,7900 etc.); BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument (MX4000 for example, MX3000, MX3005).

Description of drawings

Fig. 1 is the homology zone of DNASTAR software analysis full-length gene;

Fig. 2 is the detected result of primer sensitivity.Select for use in the full genome certain section conserved regions as standard substance.After among the figure being each copy number gradient amplification of viral RNA, the curve that fluorescence intensity changes with the reaction cycle number, wherein, X-coordinate is a cycle index, the corresponding back report fluorescence intensity (Rn) of proofreading and correct of ordinate zou representative;

Fig. 3 is the amplification of at present commercially available Chikungunya virus nucleic acid amplification detection kit.Wherein, X-axis is represented cycle index, and Y-axis is represented the corresponding back report fluorescence intensity of proofreading and correct;

Fig. 4 detects for the method set up with the present invention RNA to unknown sample.Wherein, X-axis is represented cycle index, and Y-axis is represented the corresponding back report fluorescence intensity of proofreading and correct.

Embodiment

Embodiment 1: primer sensitivity detects

1) select in the full genome of virus certain section high conserved region fragment as standard substance, with its viral RNA from 2.3 * 10 ¹⁰Copy/uL is diluted to 2.3 * 10 successively ^-1Copy/uL selects 2.3 * 10 ⁶Copy/uL to 2.3 * 10 ^-1Copy/uL copy number gradient detects, and experiment repeats 3 times, averages;

2) negative control: nuclease free water;

3) reaction system is prepared according to table 2;

4) contrast with present commercially available Chikungunya virus kit for detecting nucleic acid simultaneously.

Detected result is shown in Fig. 2, Fig. 3, table 3, table 4.

Among Fig. 2, the 1-7 curve is respectively each extent of dilution 2.3 * 10 of standard substance among the present invention ⁶Copy/uL-2.3 * 10 ⁰The curve of copy/uL amplification.

Among Fig. 3, it is 1 * 10 that the 1-3 curve is respectively each extent of dilution of test kit standard substance ³The curve of copy/uL-1copy/uL amplification.

The different copy numbers of standard substance that data shown in table 3, the table 4 are respectively used in the used positive criteria product of the present invention and the test kit are dilution

Value.Wherein,

Value result represents that for "-" this primer, probe in detecting are negative to the sample results of this gradient.The negative contrast of NTC.

As can be seen, the minimum detectability of primer among the present invention and probe in detecting positive criteria product is 2.3copy/uL from experimental result, and the minimum detectability of at present commercially available Chikungunya virus kit for detecting nucleic acid is 10copy/uL.

Table 3

Table 4

Embodiment 3: primer detects unknown sample

1) unknown sample of gathering is detected, screening datum hole Kenya virus is monitored the non-specific of primer simultaneously;

2) positive control: viral standard substance (2.3 * 10 ³Copy/uL);

3) reaction system is prepared according to table 2.

Detected result is seen Fig. 4 and table 5.

Only occurred a positive contrast of amplification curve among Fig. 4, all the other sample standard deviations do not have amplification.Reaction result is negative.The positive contrast of PTC.

Data presentation in the table 5, it is all negative to detect the unknown sample result.

Table 6

Interpretation of result:

Amplified reaction is finished after 40 circulations, the minimum detectability of primer among the present invention and probe in detecting positive criteria product is 2.3copy/uL, and the minimum detectability of at present commercially available Chikungunya virus kit for detecting nucleic acid is 10copy/uL, and primer among the present invention and probe are highly sensitive in commercially available test kit; The unknown sample detected result that collects is all negative.

The result judges:

1) when the Ct of sample to be detected (threshold value)≤36, is judged to the positive;

2) when sample 36＜Ct value to be detected＜40, need to detect again, still in 36＜Ct value＜40, then be judged to the positive as the result, otherwise negative.

Claims

1. real-time fluorescence quantitative PCR detects the method for datum hole Kenya viral nucleic acid, it is characterized in that, this method is utilized the real-time fluorescence quantitative PCR detection technique, by designing a pair of primer and probe Virus Sample is carried out the real-time quantitative detection, and designed primer and probe sequence are as follows:

2. the method for claim 1 is characterized in that, this method comprises: the acquisition of primer and probe, to the detection of datum hole Kenya viral nucleic acid; Wherein, the acquisition step of primer and probe is: (1) collects screening datum hole Kenya virus whole genome sequence, utilize information biology software to carry out the homology comparison, and at its conserved regions design primer, (2) according to the design of the nucleotide sequence in pcr amplification zone specific probe, and the specificity of login GENEBANK detection probes; Detection step to datum hole Kenya viral nucleic acid is: at first extract the nucleic acid of virus, utilize designed primer and probe that thing to be detected is carried out real-time fluorescence quantitative PCR then and detect.

3. method as claimed in claim 2 is characterized in that, described RT-PCR amplification is specially 25 μ L PCR reaction systems of the certain concentration of component of preparation.

4. the method for claim 1 is characterized in that, described sample comprises mosquito, bat, human serum and tissue, cell culture fluid, wild primates, domestic animal etc.

5. the method for claim 1 is characterized in that, in described real-time fluorescence quantitative PCR detection technique, the fluorophor that connects on the described probe comprises FAM-BHQ, FAM-TAMARA.

6. method as claimed in claim 5 is characterized in that, described RT-PCR reaction is applicable to that ABI real-time PCR system, BioRad PCR in real time detection system, Stratagene quantitative polumerase chain reaction instrument finish.