CN102534048A - Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) - Google Patents

Abstract

The invention discloses a non-diagnostic method for detecting flavivirus by a TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR). The non-diagnostic method comprises one pair of primers and the probe which are used for performing real-time quantitative detection on a sample to be detected. The invention aims to solve the problem that convenience is brought to monitoring and detecting emerging arboviruses because the flavivirus has multiple members and the detection is complicated. The defects existing in traditional and modern methods are overcome by the non-diagnostic method. The primers and the probe have the characteristics that the specificity is high, the repeatability is high, the sensitivity is high and multiple quantitation can be performed.

Description

A kind of TaqMan probe for real-time fluorescence RT-PCR detects the non-diagnostic methods of flavivirus

Technical field

The present invention relates to the non-diagnostic methods that a kind of TaqMan probe for real-time fluorescence RT-PCR detects flavivirus.

Background technology

Flaviviridae flavivirus and Togaviridae alphavirus virus, reovirus coe virus and bunyavirus coe virus all belong to arboviruses; Arboviruses is meant the beastly disease of natural focus of suffering from altogether of a kind of people that causes through the responsive vertebrates of arthropod bite of sucking blood, and is wherein maximum to the mankind's harm with flavivirus.People, animal will bring disease and death in case carried the arthropod bite of virus and infect, and when viral big area outburst, bring massive losses can for explosively economy, cause that society is panic, are the problems that various countries' public health service all might face.

At present; The main identification of means of flavivirus is that virus is separated; But because it is harsh to requirement for experiment condition, operation is complicated, technical difficulty is high and in the long serious Biosafety problem of simultaneous detection time, so promote the use of in the actual detected work again.

Use in recent years the amplification of conventional reverse transcription PCR (retro-transcript-PCR, RT-PCR) and real-time fluorescence quantitative PCR (Real-time PCR) technology brought into play vital role in the context of detection of flavivirus.But the RT-PCR that uses at present, real-time fluorescence PCR are mostly just to a certain arboviral detection.

Summary of the invention

To the problem that prior art exists, the object of the present invention is to provide a kind of quick, responsive detection and the non-diagnostic methods of screening the TaqMan probe for real-time fluorescence RT-PCR detection flavivirus of flavivirus.

For realizing above-mentioned purpose, a kind of TaqMan probe for real-time fluorescence of the present invention RT-PCR detects the non-diagnostic methods of flavivirus, it is characterized in that this non-diagnostic methods comprises:

Designed a pair of primer and probe and treated sample and originally carry out real-time quantitative and detect, primer and probe sequence are following:

Further, the fluorophor that said probe connects is: FAM-BHQl, and the fluorescence report group of fluorescent probe 5 ' end mark is FAM, the non-fluorescent quenching group of 3 ' end mark is BHQl.

Further, said this non-diagnostic methods comprises step:

1) sample is handled with lysate and RNA extract, extracts viral RNA;

2) real-time fluorescence RT-PCR reacts, and prepares 25 μ LPCR reaction systems of certain concentration of component,

Of short duration centrifugal branch installs in the PCR pipe behind the mixing;

3) testing sample is joined in the reaction system, increase;

4) collect fluorescent signal, detect the Ct value.

Further, the system component and the volume thereof of said real-time fluorescence RT-PCR reaction are following:

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

Description of drawings

Fig. 1 is an encephalitis b virus vaccine RNA amplification curve.

Fig. 2 is a yellow fever virus vaccine RNA amplification curve.

Embodiment

Below in conjunction with specific embodiment, further explain the present invention.Should be appreciated that these embodiment only to be used to the present invention is described and be not used in the scope of restriction requirement of the present invention protection that unreceipted concrete experiment condition and method in the following example are usually according to the normal condition or the condition of advising according to manufacturer.

Embodiment 1

1. sample to be checked is the encephalitis b virus vaccine, and positive control is encephalitis b virus strain RNA, and negative control is RNase-Free Water.

2. the extraction of encephalitis b virus vaccine RNA: press QIAamp Viral RNA Mini Kit working instructions and extract sample RNA.

The concrete operations step is following:

1. according to the quantity packing lysate AVL of sample, every pipe 560 μ l.

2. get 140 μ l virus liquid and join the AVL that 560 μ l branch installs, vortex concussion 15 seconds, abundant mixing, incubated at room 10 minutes.

3. every pipe adds the absolute ethyl alcohol (96%-100%) of 560 μ l respectively, shakes 15 seconds abundant mixings, and is centrifugal fast.

4. from test kit, take out the collection tube of the 2ml of band filter post, unpack marked.Get the mixed solution 630 μ ls of step in 3. and join in the collection tube centrifugal 1 minute of 8000rpm.

5. will filter post and be placed in the new 2ml collection tube, remaining mixed solution all will be drawn in the filter post centrifugal 1 minute of 8000rpm.

6. get a clean 2ml collection tube, the filter post is moved in the new collection tube, add 500 μ l elutriant AW1, centrifugal 1 minute of 8000rpm.

7. after centrifugal, abandon liquid, the filter post is placed in the new collection tube, add 500 μ l elutriant AW2, centrifugal 3 minutes of 14000rpm.Abandon liquid, once centrifugal again, to remove ethanol.

8. the filter post after centrifugal is moved on in the clean 1.5ml EP pipe, add 60 μ l lysate AVE, left standstill 2 minutes, centrifugal 1 minute of 12000rpm promptly obtains viral RNA.

3. preparation reaction system

Comprise following reagent in each reaction tubes:

System in the 1.5mL centrifuge tube outside 4 removing template RNA of preparation, of short duration centrifugal branch installs in 3 PCR reaction tubess behind the mixing, adds negative control, encephalitis b virus vaccine RNA, positive control respectively.

Last machine increases amplification condition: the standard amplification condition that adopts this area under following condition.

45 ℃ 15 minutes

95 ℃ 10 minutes

4. the collection fluorescent signal detects the Ct value

5. the result judges: according to positive control, negative control Ct value is judged.When the Ct value is judged as the positive smaller or equal to 37 the time.Be illustrated in figure 1 as encephalitis b virus vaccine RNA amplification curve, wherein: red positive contrast Ct value 19.2, green is an encephalitis b virus vaccine RNA Ct value 23, blue negative control test is less than the Ct value.

Embodiment 2

1. the extraction of yellow fever virus vaccine RNA: extract test kit with QIAamp Viral RNA, the by specification operation.

2. preparation reaction system

Comprise following reagent in each reaction tubes:

3. go up machine, under following condition, increase amplification condition: the standard amplification condition that adopts this area.

45 ℃ 15 minutes

95 ℃ 10 minutes

4. the collection fluorescent signal detects the Ct value

5. the result judges.Be illustrated in figure 2 as yellow fever virus vaccine RNA amplification curve, wherein: red positive contrast Ct value 19.2, brown is yellow fever virus vaccine RNA Ct value 25, blue negative control test is less than the Ct value.

Above-mentioned virus is extracted from test kit, and detection method disclosed by the invention is not to be object with human body and animal, just in order to detect whether have virus, is not to be diagnosed as purpose, not belong to the diagnostic method of disease.

Only if specifically defined, it is known term in the relevant technologies field that the present invention describes used term.The chemical symbol of standard and dummy suffix notation can exchange with its full name and use.

Only if special indicating, the present invention uses but clearly sets forth or simple technology and the method for setting forth is meant normally used technology in present technique field and method, can carry out according to technology well known in the art and method.The use of test kit is to carry out according to the specification sheets that manufacturers or supplier provide.

Claims (4)

1. a TaqMan probe for real-time fluorescence RT-PCR detects the non-diagnostic methods of flavivirus, it is characterized in that this non-diagnostic methods comprises:

Designed a pair of primer and probe and treated sample and originally carry out real-time quantitative and detect, primer and probe sequence are:

2. non-diagnostic methods as claimed in claim 1 is characterized in that, the fluorophor that said probe connects is: FAM-BHQl, and the fluorescence report group of fluorescent probe 5 ' end mark is FAM, the non-fluorescent quenching group of 3 ' end mark is BHQl.

3. non-diagnostic methods as claimed in claim 1 is characterized in that, said this non-diagnostic methods comprises step:

1) sample is handled with lysate and RNA extract, extracts viral RNA;

2) real-time fluorescence RT-PCR reacts, and prepares 25 μ LPCR reaction systems of certain concentration of component, and of short duration centrifugal branch installs in the PCR pipe behind the mixing;

3) testing sample is joined in the reaction system, increase;

4) collect fluorescent signal, detect the Ct value.

4. non-diagnostic methods as claimed in claim 3 is characterized in that, the system component and the volume thereof of said real-time fluorescence RT-PCR reaction are following:

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

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