CN109355436A - A kind of RT-LAMP Primer composition and its application detecting PRRSV - Google Patents
A kind of RT-LAMP Primer composition and its application detecting PRRSV Download PDFInfo
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Abstract
The present invention provides a kind of RT-LAMP Primer compositions and its application for detecting PRRSV, the Primer composition includes: outer primer F3 and B3, inner primer FIP and BIP and ring primer LF and LB, the outer primer sequence is as shown in SEQ ID No.1 and SEQ ID No.2, the inner primer sequence is as shown in SEQ ID No.3 and SEQ ID No.4, and the ring primer sequence is as shown in SEQ ID No.5 and SEQ ID No.6.The primer detects porcine reproductive and respiratory syndrome virus using RT-LAMP method, has the characteristics that quick, convenient, sensitive, efficient, high specificity.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of RT-LAMP Primer composition for detecting PRRSV
And its application.
Background technique
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS it) is also blue otopathy, is by porcine reproductive and respiratory syndrome virus (Porcine reproductive and
Respiratory syndrome virus, PRRSV) caused by the great epidemic disease of a boar.Porcine reproductive and respiratory syndrome virus
(PRRSV) full-length genome is about 15Kb, is a kind of normal chain picornavirus, with equine arteritis virus (EAV), rush lactic dehydrogenase
Viral (LDV) and SHF virus (SHFV) belong to Arteriviridae.The disease is in 1987 for the first time in the north card in the U.S.
The states such as Luo Na, Iowa are broken out, and are spread rapidly in the whole America.It is annual because economic loss caused by PRRS is up to 5.6 hundred million in the U.S.
~6.6 hundred million dollars.In China, since 1996 are separated to PRRSV for the first time, domestic major pig farm has been spread all over quickly, it is main
Clinical symptoms be piglet expiratory dyspnea, hyperpyrexia do not move back, the breeding difficultys such as rubefaction and Sow abortion, stillborn foetus, give
The pig breeding industry of state has brought tremendous economic losses, and causes industry and widely pay close attention to.Currently, the diagnosis to the disease is mainly
The laboratories coherent detections such as virus purification, ELISA or RT-PCR are carried out after carrying out pathological material of disease acquisition to doubtful sick pig, but these are diagnosed
Time-consuming for method, at high cost, the high requirements on the equipment, is unfavorable for epidemic disease scene simplicity, fast and accurately makes a definite diagnosis and apply, thus
Affect the prevention and control of the disease.
Loop-mediated isothermal amplification technology (Loop Mediated Isothermal Amplification, LAMP) is a kind of
One kind that assistance under the conditions of isothermal reaction using specific primer and amplification enzyme expands template in cycles is quick
Diagnostic techniques.The technology is with its high sensitivity, the features such as high specificity, the reaction time is short, easy to operate, makes it quickly in prevalence
The fields such as sick Hygienic monitoring on hands of childhood, medical diagnosis on disease, animal pathogenic analyte detection, food safety detection are widely used.Reverse transcription loop
Mediated constant temperature nucleic acid amplification (RT-LAMP) is that suitable reverse transcriptase and Bst archaeal dna polymerase is added in one kind in former reaction solution
The RNA virus Fast Detection Technique achieved that.In recent years, which has been widely used in pathogen detection, such as swine flu
The thin circovirus virus of virus, foot and mouth disease virus, swine fever virus, porcine pseudorabies virus, Porcine epidemic diarrhea virus, pig, Ebola virus
Deng all establishing this technology in succession.The present invention is based on RT-LAMP technical principles, design spy for the ORF5 genetic fragment of PRRSV
Specific primer, it is intended to establish the rapid detection method for being suitable for primary diagnosis porcine reproductive and respiratory syndrome virus.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of specific RT-LAMP primer combination for detecting PRRSV
Object and its RT-LAMP detection reagent box and RT-LAMP (ring mediated reverse transcription isothermal duplication) detection method, this method is quickly, just
Prompt, sensitive, efficient, high specificity, overcome current viral diagnosis it is cumbersome, at high cost, with duration the problems such as.
In order to reach above-mentioned technical effect, the present invention is realized especially by following technical scheme:
A kind of RT-LAMP Primer composition detecting PRRSV, the Primer composition includes: outer primer F3 and B3, interior
Primers F IP and BIP and ring primer LF and LB, the outer primer sequence as shown in SEQ ID No.1 and SEQ ID No.2,
The inner primer sequence is as shown in SEQ ID No.3 and SEQ ID No.4, the ring primer sequence such as SEQ ID No.5
With shown in SEQ ID No.6.
Preferably, the primer further includes by F3, B3, FIP, BIP, LF and LB primer sequence by one or several cores
Have and above-mentioned sequence DNA molecular with the same function after the substitution and/or deletion and/or addition of thuja acid.
The present invention provides above-mentioned Primer compositions in RT-LAMP method to detect porcine reproductive and respiratory syndrome disease
The application of poison.
The reaction condition of the RT-LAMP is 95 DEG C of 5min high-temperature denaturations；65 DEG C of warm bath 45min；80 DEG C of 2min are terminated
Reaction.
The present invention also provides above-mentioned Primer compositions in preparation detection porcine reproductive and respiratory syndrome virus kit
Application.The preparation method of the kit includes the steps that individually packing each primer.
It is a kind of for detecting the kit of porcine reproductive and respiratory syndrome virus, the kit passes through the side RT-LAMP
Method is detected, and the kit includes primer shown in No.1~6 SEQ ID.
The invention has the benefit that
The present invention designs six primers for PRRSV, can be fast implemented using RT-LAMP technology comprehensive to pig breeding and breathing
The detection of simulator sickness virus, tests prove that the technology of the present invention time-consuming is short, whole constant temperature is not necessarily to large-scale precision instrument, can raise pigs
Field or small room are completed.Reduce the possibility of cause of disease mass degradation in sample, whole process is easy to operate, as a result detection side
Just, so operating without extremely professional personnel, large-scale promotion is suitble to use.
Detailed description of the invention
Fig. 1 is the electrophoretogram of 2 ring primer sets of embodiment of the present invention detection PRRSV；M:DL2000DNA marker；1: often
Advise primer；2: primer containing Loop；3: negative control；
Fig. 2 is the specificity experiments electrophoretogram of primer sets of the present invention；M, DNA Marker；1-5, respectively CSFV, PCV-
2,PRRSV,PPV,PRV；
Fig. 3 is the sensitivity experiments electrophoretogram of primer sets of the present invention；A:M, DNA Marker；1-5, respectively 10-5、10-4、10-3、10-2、10-1The template of dilution；B:M, DNA Marker；1-5, respectively 10-1、10-2、10-3、10-4、10-5It is dilute
The template for degree of releasing；
Fig. 4 is the clinical application verifying electrophoretogram of primer sets of the present invention；A:LAMP electrophoretogram, b:RT-PCR electrophoretogram, M:
Super DNA marker, 1-10: doubtful clinic pathological material of disease.
Specific embodiment
Below in conjunction with specific embodiment of the present invention, technical solution of the present invention is clearly and completely described, is shown
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Virus and pathological material of disease: porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus (CSFV), porcine pseudorabies virus
(PRV), pig parvoviral (PPV), porcine circovirus 2 type (PCV2) are protected by animal applications technical research institute of Mianyang Teachers College
It deposits；The clinic pathological material of disease such as serum, lymph node is provided by Sichuan cavalry man of great strength group Feng Guang moral laboratory.
Main agents: 10 × Thermopol reaction buffer, Bst archaeal dna polymerase, MgSO4Purchased from knob Great Britain biology skill
Art (Beijing) Co., Ltd；dNTP Mixture,DNA Marker,Mini BEST Viral RNA/DNA Extraction
Kit Ver.5.0、PrimeScript TM One Step RT-PCR Kit Ver.2、 PrimeScript TM 1st
Strand cDNA Synthesis Kit is purchased from Takara company；Agarose is purchased from Invitrogen company；SYBR Green
I is purchased from Xiamen Zeesan Biotech Co., Ltd..
1 RNA of embodiment extraction is synthesized with virus cDNA
Use micro Viral nucleic acid extraction reagent box TaKaRa Mini BEST Viral RNA/DNA Extraction
The nucleic acid extraction of Kit Ver.5.0 progress PRRSV.According to the kit specification, viral lived vaccine nucleic acid RNA is extracted, is protected
Be stored in -80 DEG C it is spare or use immediately.
It is template using the PRRSV nucleic acid RNA mentioned above taken, using reverse transcription reagent box (PrimeScript RT
Mater Mix (RR036A)) carry out reverse transcription, corresponding first DNA chain of synthesis viral nucleic acid, i.e. cDNA.Reverse transcription system
It is shown in Table 1.
1 reverse transcription system of table
The design and synthesis of 2 primer of embodiment
Referring to PRRSV gene order (accession number: EU860248) design primer of GenBank, 6 LAMP primers are designed,
Including two inner primers FIP and BIP, two outer primers F3 and B3, two ring primers Fs and B, primer information are shown in Table 2.
2 LAMP primer sequence of table
A pair of of specificity Loop primer (Loop-B/Loop-F) is designed, Loop primer can be combined and be formed in the amplification reaction
Loop-stem structure on, accelerate the formation of ring, be more advantageous to the rapid amplifying of aim sequence, improve amplification efficiency.Therefore, it is reacting
Loop primer is added in system and is more advantageous to the progress of reaction, and shortens the reaction time to a certain extent, improves reaction efficiency.
Loop primer is added in reaction system and carries out LAMP reaction, as a result as shown in Figure 1.As seen from the figure, after Loop primer being added, reaction
Expanding effect is more preferably.Therefore the Loop primer that this experiment is established is successful.
The foundation of 3 PRRSV RT-LAMP detection method of embodiment
The foundation of reaction system is shown in Table 3.When preparing LAMP reaction system, it should be noted that: Bst DNA polymerase is being added
Before, it needs that the mixed liquor of other prepared reactants is denaturalized DNA, is immediately placed on ice in 95 DEG C of warm bath 5min
It is cooling, a certain amount of Bst archaeal dna polymerase is added after cooling, is uniformly mixed.Reaction condition are as follows: 95 DEG C of 5min high-temperature denaturations；
65 DEG C of warm bath 45min；80 DEG C of 2min terminate reaction.Reaction product obtained by 4 μ L is taken after reaction terminating, carries out agarose gel electrophoresis
(EB dyeing), gel strength 1.5%, attached gel imaging system show result.
The LAMP reaction system that table 3 is tentatively established
The specificity of 4 LAMP of embodiment reaction primer
Five kinds of viral nucleic acids of PRRSV, CSFV, PCV-2, PPV and PRV are extracted, it is comprehensive according to the pig breeding and breathing established
Simulator sickness virus RT-LAMP detection method carries out specific test and tradition RT-PCR is tested, and verifies the PRRSV RT-LAMP of foundation
The specificity of detection method.
Using 5 kinds of viral nucleic acids of PRRSV, CSFV, PCV-2, PPV and PRV as the special of template detection RT-LAMP system
Property, as a result as shown in Figure 2.As shown in Figure 2, porcine reproductive and respiratory syndrome virus RT-LAMP detection method can only expand pig breeding
With breath syndrome virus nucleic acid and cannot detect other non-targeted nucleic acid, so the RT-LAMP established have it is special well
It is anisotropic.
The sensitivity of 5 PRRSV RT-LAMP of embodiment is tested
Continuous 10 times of gradient dilutions are carried out to sample with deionized water, are reacted and tradition RT- using the RT-LAMP after optimization
PCR reaction, verifies the sensitivity of the PRRSV RT-LAMP detection method of foundation.
With 10-1-10-5The porcine reproductive and respiratory syndrome virus nucleic acid of dilution be template carry out respectively RT-LAMP and
RT-PCR reaction, analyzes RT-LAMP detection sensitivity, as a result such as Fig. 3.As shown in Fig. 3 a, RT-LAMP be can detecte to 10-5It is dense
Nucleic acid is spent, and it is as shown in Figure 3b, and RT-PCR is only able to detect 10-3Concentration nucleic acid.Identical result is all obtained by test of many times.
So it is 100 times high to establish porcine reproductive respiratory syndrome virus RT-LAMP detection method ratio RT-PCR sensitivity.
6 PRRSV RT-LAMP clinical application of embodiment verifying
10 parts of doubtful clinical pathological material of diseases (serum, lymph node etc.) of acquisition are examined in this planning of experiments using LAMP detection method
It surveys, and at the same time carrying out traditional PCR method detection, is compared detection, verify the degree of conformity of the two.
Using the method for control, the verifying for carrying out clinical application is reacted LAMP.Simultaneously by LAMP reaction and conventional PCR
It carries out, 10 parts of doubtful clinical pathological material of diseases (serum, lymph node etc.) of acquisition is detected, as a result as shown in Figure 4.As shown in Figure 4,
All show in this batch of pathological material of disease there are 4 positive samples in the result of LAMP detection and PCR detection, therefore the degree of conformity of the two is
100%.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Sequence table
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Claims (7)
1. a kind of RT-LAMP Primer composition for detecting PRRSV, which is characterized in that the Primer composition includes: outer primer F3
With B3, inner primer FIP and BIP and ring primer LF and LB, the outer primer sequence such as SEQ ID No.1 and SEQ ID
Shown in No.2, the inner primer sequence is as shown in SEQ ID No.3 and SEQ ID No.4, the ring primer sequence such as SEQ
Shown in ID No.5 and SEQ ID No.6.
2. a kind of RT-LAMP Primer composition for detecting PRRSV according to claim 1, which is characterized in that described draws
Object further include by F3, B3, FIP, BIP, LF and LB primer sequence by one or several nucleotide substitution and/or missing and/
Or have and above-mentioned sequence DNA molecular with the same function after addition.
3. Primer composition described in claim 1 is in RT-LAMP method to detect porcine reproductive and respiratory syndrome virus
Application.
4. application according to claim 3, which is characterized in that the reaction condition of the RT-LAMP is 95 DEG C of 5min high
Temperature denaturation；65 DEG C of warm bath 45min；80 DEG C of 2min terminate reaction.
5. Primer composition described in claim 1 answering in preparation detection porcine reproductive and respiratory syndrome virus kit
With.
6. a kind of for detecting the kit of porcine reproductive and respiratory syndrome virus, which is characterized in that the kit includes
Primer shown in No.1~6 SEQ ID.
7. kit according to claim 6, which is characterized in that the preparation method of the kit includes by each primer
Individually the step of packaging.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235317A (en) * | 2020-03-26 | 2020-06-05 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting PRRSV (porcine reproductive and respiratory syndrome Virus) and PCV (porcine circovirus Virus) |
| CN113846092A (en) * | 2020-08-06 | 2021-12-28 | 中国农业科学院深圳农业基因组研究所 | Special primer combination and detection kit for simply determining PRRSV (porcine reproductive and respiratory syndrome virus) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235317A (en) * | 2020-03-26 | 2020-06-05 | 广西壮族自治区兽医研究所 | Primer composition, kit and method for detecting PRRSV (porcine reproductive and respiratory syndrome Virus) and PCV (porcine circovirus Virus) |
| CN113846092A (en) * | 2020-08-06 | 2021-12-28 | 中国农业科学院深圳农业基因组研究所 | Special primer combination and detection kit for simply determining PRRSV (porcine reproductive and respiratory syndrome virus) |
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