CN110387437A - A kind of detection mouse encephalomyelitis virus RT-RPA amplification method and its kit - Google Patents
A kind of detection mouse encephalomyelitis virus RT-RPA amplification method and its kit Download PDFInfo
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Abstract
The invention discloses a kind of mouse encephalomyelitis virus RT-RPA primer sets, kit and its detection methods, real-time detection mouse encephalomyelitis virus may be implemented, reaction can be completed within 20 minutes under constant temperature conditions, has the characteristics that easy to operate, high specificity, high sensitivity.Reaction detection sensitivity of the invention reaches 1 × 101Copies provides technical support for quickly detection, there is high application value in the pathogenic microorganism examination.
Description
Technical field
The present invention relates to the pathogenic microorganism examination technical research fields, and in particular to a kind of quickly detection mouse encephalomyelitis
Viral RT-RPA method and its kit.
Background technique
Mouse encephalomyelitis virus (Theiler ' s murine encephalomyelitis virus, TMEV) is experiment
Common one of viral cause of disease in animal mouse population.The virus belongs to microRNA Viraceae, cardiovirus
(Cardiovirus).TMEV genome includes a sub-thread strand RNA, length about 8100bp.In mouse group, TMEV infection
It is a commonplace disease, studies have reported that its infection rate is 8%~35%.It is most of after the mouse infection virus
It is in subclinical infection without visible clinical symptoms；But infecting mouse can be caused to occur to be immunoreacted and then cause immunologic derangement etc.
Pathological phenomenon, this not only causes actual bodily harm to mouse itself, but also can interfere life science as a result, therefore TMEV
It is the pathogen that SPF grades of mouse need to exclude in national standard.Studies have found that, TMEV is seeded in the big of rat in recent years
Brain can cause it that visible clinical symptoms, such as hind limb paralysis and weight loss symptom occur.
Currently, TMEV diagnostic method is broadly divided into Serologic detection and pathogeny detection.Serologic detection mainly has enzyme-linked
Immunosorbent adsorption test (ELISA), immunofluorescent test (IFA) etc..Since immunodeficiency animal such as nude mice or gene change
Mouse etc. is made, it cannot be with the immune response as normal mouse generation；Virus infection early stage body not yet generates antibody, the stage
False negative result certainly will be led to by carrying out antibody test；And can not to the environment that may pollute of virus such as mouse cage tool and padding into
Row detection, therefore Serologic detection is applied with limitation.Pathogeny detection method especially RT-PCR is easy to operate, sensitive
Degree is high, has become an important molecule technological means of virus monitor.But regular-PCR or quantitative fluorescent PCR, all extremely
A small the reaction time is needed less, and needs expensive instrument, and many grass-roots units and remote districts can not undertake.Cause
This, a kind of reagent for the recombinase polymeric enzymatic amplification (RPA) that can detect mouse encephalomyelitis virus be at present needed for.
Summary of the invention
The primary purpose of the present invention is that providing the primer sets and probe of a pair of of detection mouse encephalomyelitis virus；
Another object of the present invention is to provide a kind of kits of mouse encephalomyelitis virus；
A further object of the present invention is to provide a kind of detection methods of mouse encephalomyelitis virus.
The technical solution used in the present invention is:
A kind of primer sets for the RT-RPA detecting mouse encephalomyelitis virus, nucleotide sequence are as follows:
TMEV-F:5 '-TTCCTATGGAGATGACCTATTAATTGGAAC-3 '；
TMEV-R:5 '-TCAGAGGAAAGGTGGTGGTCTTGTTGGCAG-3 '.
A kind of fluorescence probe for the RT-RPA detecting mouse encephalomyelitis virus, nucleotide sequence are as follows:
TAATCTTATAACCGAAGGGGGCTAATCTTT-THF-TTTAACAAGATTAAA。
A kind of detection kit for the RT-RPA detecting mouse encephalomyelitis virus, the kit contain described above draw
Object group.
Further, probe described above is also contained in the kit.
Further, the also recombinase polymeric enzymatic amplification reagent containing target gene in the kit.
A kind of detection method of the RT-RPA of mouse encephalomyelitis virus comprising the steps of:
1) sample RNA is extracted；
2) using the RNA of extraction as template, primer sets TMEV-F, TMEV-R described above and described above are used
Probe carry out RT-RPA amplified reaction obtain amplified production；
3) tracing analysis is carried out to amplified production, determines in sample whether contain mouse encephalomyelitis virus.
Further, 10 μM are added into the 50ul amplification system of RPA amplifing reagent kit specification recommendation response
Primer
Each 2 μ L, 10 μM of 0.6 μ L of probe, 2 μ L RNA.
Further, the amplification reaction condition of step 2) are as follows: 39 DEG C of isothermal reaction 20min.
The beneficial effects of the present invention are:
Kit provided by the invention can be used for quickly detecting mouse encephalomyelitis virus, and the reaction time only needs 20 points
Clock, and can real-time monitoring amplified production under constant temperature conditions, have that accuracy is high, specificity is good, the high feature of repeatability,
And can be reacted using light fluorescence detector, be conducive to promote and apply in vast grass-roots unit and clinical practice.
Detailed description of the invention
Fig. 1 is the specific amplification curve figure of mouse encephalomyelitis virus RT-RPA method of the present invention；
Fig. 2 is the sensitive amplification curve graph of mouse encephalomyelitis virus RT-RPA of the present invention, from left to right 1.0 × 106、
1.0×105、1.0×104、1.0×103、1.0×102、1.0×101, 1.0 × 100copies/ μ l standard items amplification knot
Fruit；
Fig. 3 is the clinical sample confirmatory experiment result figure of mouse encephalomyelitis virus RT-RPA of the present invention.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The design of 1 specific primer of embodiment
The present invention analyzes its sequence portion the most conservative according to the mouse encephalomyelitis virus genome sequence published
Divide the design for carrying out primer；It carries out preliminary screening to designed primer, obtains one group suitable for the sensitivity of RPA and special
The preferable primer pair of property, nucleotide sequence are as follows:
TMEV-F:5 '-TTCCTATGGAGATGACCTATTAATTGGAAC-3 ' (SEQ.ID:NO.1)；
TMEV-R:5 '-TCAGAGGAAAGGTGGTGGTCTTGTTGGCAG-3 ' (SEQ.ID:NO.2)；
Expanding fragment length is 104bp；
Probe sequence is as follows:
TAATCTTATAACCGAAGGGGGCTAATCT(FAM-dT)T(THF)T(BHQ-dT)TAACAAGATTAAA(3’
spacer)(SEQ.ID:NO.3)。
Embodiment 2 detects the foundation of the RT-RPA kit of TMEV
Kit includes following components:
(1) primer sets and probe designed by embodiment 1；
(2) recombinase of target gene amplification polymerize enzymatic reagent, which is purchased from Hangzhou Zhong Ce company；
(3) RT-RPA amplification reaction system.
The preparation of 3 positive criteria template of embodiment, RT-RPA detection method
1) preparation of RNA standard form
It extracts mouse encephalomyelitis virus RNA, One Step is used with conservative region design primer (primer sequence is as follows)
RT-PCR Kit Ver.2 kit (Takara company) carries out RT-PCR and obtains the conserved sequence that length is 800bp.Primer
Nucleotide sequence are as follows:
Upstream primer F:TGGAACTGACCCGGATGTTG (SEQ.ID:NO.4)
Downstream primer R:GCGTACACCCTAGTGGCTTC (SEQ.ID:NO.5)
Reaction system are as follows:
RT-PCR response procedures are as follows: 50 DEG C of reverse transcription 30min；94 DEG C of initial denaturation 2min；94 DEG C of denaturation 30s, 50 DEG C of annealing
30s, 72 DEG C of extension 40s are recycled 35 times；Last 72 DEG C of extensions 5min.Pcr amplification product is cut through 2% agarose gel electrophoresis
Target fragment is connect with pGEM-T Easy carrier after purification, is transformed into Escherichia coli, and picking single colonie culture extracts plasmid simultaneously
Double digestion is identified that positive plasmid pGEM-TMEV is sequenced.
It will be purified after pGEM-TMEV plasmid linearization, with in-vitro transcription kit RibomaxTM Large Scale RNA
Production Systems (Promega company) illustrates the transcription for carrying out target gene, obtains cell-free transcription folder TMEV-RNA,
And according to the RNA concentration calculation copy number of spectrophotometric determination.
2) RT-RPA amplification (the recombinase polymerization enzymatic reagent of target gene amplification is purchased from Hangzhou Zhong Ce company)
10 μM of RPA primer is added into the 50uL amplification system of RPA amplifing reagent kit specification recommendation response
(TMEV-F, TMEV-R) each 2 μ L, 10 μM of 0.6 μ L of probe, 2 μ L RNA.
Amplified reaction program are as follows: 39 DEG C of reaction 20min.
3) interpretation of result
The reaction tube that configuration is completed is placed in fluorescence detector, the fluorescence signal detected according to instrument is seen whether
Generate fluorescence signal curve.As a result judgment criteria is as follows:
In 20 minutes, there is apparent amplification curve to generate, be judged as positive sample；There is no apparent amplification curve raw
At being judged as negative sample.
4 RT-RPA detection method specificity experiments of embodiment
Mouse hepatitis virus (MHV), mouse norovirus (MNV), sendai virus (SV), mouse pneumonia virus are extracted respectively
(PVM), the viral genome of mouse encephalomyelitis virus (TMEV) is as template, ddH2O carries out RT-RPA inspection as a control group
It surveys.As shown in Figure 1, only TMEV has fluorescent amplification curve generation, and tetra- viruses of MHV, MNV, SV and PVM and control group do not have
Amplification curve is in smooth shape.Illustrate that detection method specificity of the invention is good.
5 RT-RPA detection method sensitivity experiment of embodiment
The RNA template that will be prepared in embodiment 3 measures concentration and is scaled the copy number of target gene, and carries out 10
It dilutes again, obtains 100To 106Copies/ μ l totally 7 dilutions use above-mentioned reaction system and reaction condition to carry out RT-RPA spirit
The detection of sensitivity, while setting up negative control group.As a result as shown in Fig. 2, to 101When copies reacts, there is fluorescent amplification curve,
It detects mouse encephalomyelitis virus, illustrates that the sensitivity of this method is preferable.The present invention detects limit and has reached 101copies。
The RT-RPA of 6 clinical sample of embodiment detects verifying
1, the preparation of RNA
14 Mice brain tissues through experimental infection TMEV are acquired, extract geneome RNA after preparation homogenate.
2, RT-RPA amplification (the recombinase polymerization enzymatic reagent of target gene amplification is purchased from Hangzhou Zhong Ce company)
Suggest according to the manufacturer of RT-RPA reagent, expands body to the 50ul of RPA amplification kit specification recommendation response
The concentration that 10 μM of RPA primer (TMEV-F, TMEV-R) each 2 μ L, 0.6 μ L are added in system is 10 μM of probes, 2 μ L RNA.
Amplified reaction program are as follows: 39 DEG C of reaction 20min.
3. for RT-RPA as a result, it has been found that 1-12 sample is the positive, 13 and No. 14 are feminine gender experimental result is shown in Fig. 3；It uses
Conventional RT-PCR verifies sample, and discovery 1-11 sample is the positive；Sequencing identification discovery 1-11 sample is TMEV,
As a result, it has been found that the accuracy of result of the present invention is up to 100%, and higher than conventional RT-PCR sensibility.
The above is the preferable embodiment of the present invention, but embodiments of the present invention are not by the limit of above-described embodiment
System, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>a kind of detection mouse encephalomyelitis virus RT-RPA amplification method and its kit
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213>artificial primer
<400> 1
ttcctatgga gatgacctat taattggaac 30
<210> 2
<211> 30
<212> DNA
<213>artificial primer
<400> 2
tcagaggaaa ggtggtggtc ttgttggcag 30
<210> 3
<211> 44
<212> DNA
<213>artificial primer
<400> 3
aatcttataa ccgaaggggg ctaatctttt ttaacaagat taaa 44
<210> 4
<211> 20
<212> DNA
<213>artificial primer
<400> 4
tggaactgac ccggatgttg 20
<210> 5
<211> 20
<212> DNA
<213>artificial primer
<400> 5
gcgtacaccc tagtggcttc 20
Claims (8)
1. a kind of primer sets for the RT-RPA for detecting mouse encephalomyelitis virus, which is characterized in that the following institute of its nucleotide sequence
Show:
TMEV-F:5 '-TTCCTATGGAGATGACCTATTAATTGGAAC-3 '；
TMEV-R:5 '-TCAGAGGAAAGGTGGTGGTCTTGTTGGCAG-3 '.
2. a kind of probe for detecting mouse encephalomyelitis virus, which is characterized in that its nucleotide sequence is as follows:
TAATCTTATAACCGAAGGGGGCTAATCTTT-THF-TTTAACAAGATTAAA。
3. a kind of detection kit for the RT-RPA for detecting mouse encephalomyelitis virus, which is characterized in that the kit, which contains, has the right
Benefit require 1 described in primer sets.
4. kit according to claim 3, which is characterized in that also contain spy as claimed in claim 2 in the kit
Needle.
5. kit according to claim 3, which is characterized in that also the recombinase containing target gene is poly- in the kit
Synthase amplifing reagent.
6. a kind of detection method of the RT-RPA of mouse encephalomyelitis virus, which is characterized in that comprise the steps of:
1) sample RNA is extracted；
2) using the RNA of extraction as template, primer sets TMEV-F, TMEV-R and claim described in claim 1 are used
Probe described in 2 carries out RT-RPA amplified reaction and obtains amplified production；
3) tracing analysis is carried out to amplified production, determines in sample whether contain mouse encephalomyelitis virus.
7. according to the method described in claim 6, it is characterized in that, expanding to the 50ul of RPA amplifing reagent specification recommendation response
10 μM of primer each 2 μ L, 10 μM of 0.6 μ L of probe, 2 μ L RNA are added in increasing system.
8. according to the method described in claim 6, it is characterized in that, the amplification reaction condition of step 2) are as follows: 39 DEG C of isothermal reactions
20min。
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