CN110273025A - A kind of Alon mountain virus PCR detection primer group, kit and application - Google Patents
A kind of Alon mountain virus PCR detection primer group, kit and application Download PDFInfo
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- CN110273025A CN110273025A CN201910533839.4A CN201910533839A CN110273025A CN 110273025 A CN110273025 A CN 110273025A CN 201910533839 A CN201910533839 A CN 201910533839A CN 110273025 A CN110273025 A CN 110273025A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
Abstract
The invention belongs to viruses molecule detection technique fields, and in particular to a kind of Alon mountain virus PCR detection primer group, kit and application.The nucleotide sequence of the Alon mountain virus PCR detection primer group is as shown in No.1~3 SEQ ID, and the present invention also provides a kind of kits comprising above-mentioned primer sets.Alon mountain virus PCR detection primer group provided by the invention and kit energy are accurate, quick, sensitivity, specifically detect Alon mountain virus, with rapidly and efficiently, the convenient, high specific of operation, high sensitivity, identification is simple, at low cost, do not need expensive instrument, is suitble to the beneficial effects such as on-site test, foundation for Alon mountain method for detecting virus is laid a good foundation, and has certain application value and realistic meaning.
Description
Technical field
The invention belongs to viruses molecule detection technique fields, and in particular to a kind of Alon mountain virus PCR detection primer group, examination
Agent box and application.
Background technique
Alon mountain virus (Alongshan virus, ALSV) is a kind of newfound segmented flavivirus, is mainly passed through
Tick bites propagation, infected cattle, sheep, people etc..ALSV is upper closely related in evolution with Jingmen ixodism poison, and genome is all divided into four pieces
Section, overall length 11350bp, S1 piece segment length's 2995bp encoding flaviviral NS5 sample NSP1 albumen；S3 piece segment length's 2811bp encoding flaviviral
NS2b-NS3 sample albumen NSP2；S2 piece segment length 2806bp encodes glycoprotein VP1；S4 piece segment length 2738bp, encoding nuclear proteins VP2
With glycoprotein V P3.The molecular biology for detection of Alon mountain virus is blank at present, this research is proposed to stand a kind of detection Ah
The method of Longshan virus.
Summary of the invention
For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing a kind of Alon mountain virus
PCR detection primer group.
Another object of the present invention is to provide the applications of above-mentioned Alon mountain virus PCR detection primer group.
A further object of the present invention is to provide a kind of Alon mountain virus PCR detection kit, which includes above-mentioned
Alon mountain virus PCR detection primer group.
Fourth object of the present invention is to provide the application of above-mentioned Alon mountain virus PCR detection kit.
A kind of method that of the invention the 5th is designed to provide PCR detection Alon mountain virus.
The purpose of the invention is achieved by the following technical solution:
A kind of Alon mountain virus PCR detection primer group, include primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2,
Its nucleotide sequence is as follows:
Primer ALSV-F1:5 '-GTVTTYGTGCCRGGHCTGAC-3 '；
Primer ALSV-R1:5 '-GTGGABGGRGTTATBAYYCCYTTNG-3 '；
Primer ALSV-R2:5 '-TGGCABGTRTCRAAMACWGCRTC-3 '；
Wherein, V, Y, R, H, B, N, M and W indicate degeneracy base code, V=A/G/C；Y=C/T；R=A/G；H=A/C/
T；B=G/C/T；N=A/T/C/G；M=A/C；W=A/T；
Application of the Alon mountain virus PCR detection primer group in the viral diagnosis of Alon mountain；
Application of the Alon mountain virus PCR detection primer group in the viral diagnosis of Alon mountain does not include the diagnosis of disease
Purpose；
A kind of Alon mountain virus PCR detection kit includes above-mentioned Alon mountain virus PCR detection primer group；
The Alon mountain virus PCR detection kit, preferably comprises 10 μM of ALSV-F1,10 μM of ALSV-R1, and 10 μM
ALSV-R2,10mM dNTP, 10 × Reaction Buffer, archaeal dna polymerase and deionized water；
Application of the Alon mountain virus PCR detection kit in the viral diagnosis of Alon mountain；
Application of the Alon mountain virus PCR detection kit in the viral diagnosis of Alon mountain does not include the diagnosis of disease
Purpose；
A kind of method of PCR detection Alon mountain virus, comprises the following steps:
(1) it extracts the RNA of sample to be tested and reverse transcription is cDNA；
(2) PCR system is prepared, using cDNA made from step (1) as template, with above-mentioned Alon mountain virus PCR detection primer
Group or primer ALSV-F1 in above-mentioned Alon mountain virus PCR detection kit and primer ALSV-R1 are amplimer, carry out the
One wheel PCR amplification；
(3) PCR system is prepared, the first round PCR product obtained using step (2) amplification is template, with above-mentioned Alon mountain disease
Malicious PCR detection primer group or primer ALSV-F1 in above-mentioned Alon mountain virus PCR detection kit and primer ALSV-R2 are to expand
Increase primer, carries out the second wheel PCR amplification；
(4) the second wheel PCR product for obtaining step (3) amplification carries out agarose gel electrophoresis detection, goes out at 224bp
Existing purpose band, then be determined as the positive, do not occur purpose band at 224bp, be then determined as feminine gender；
PCR system described in step (2), preferably comprises following component:
PCR system described in step (3), preferably comprises following component:
The reaction condition of PCR amplification described in step (2) and step (3) is preferred are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation
30s, 50 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 10 recycle；94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extension 30s, 25
A circulation；72 DEG C of extension 10min；
It should be pointed out that detection of the invention is for the detection to vitro samples, the direct result of detection is viral
Magnitude, and not diagnostic result, the direct purpose of the present invention are not diagnosis, the application and method of primer of the invention, kit
It is not belonging to diagnostic method.
The present invention has the following advantages and effects with respect to the prior art:
(1) the present invention provides a kind of Alon mountain virus PCR detection primer group, which can be with specific amplification Alon
Mountain conserved viral sequences provide technical support to the Testing and appraisal of Alon mountain virus.
(2) present invention devises two groups of different Alon mountain virus PCR detection primer groups, and compares its detection efficiency, ties
Fruit shows that primer sets provided by the present invention have the advantages that accurate energy, quick, sensitivity, specifically detect Alon mountain virus,
Foundation for Alon mountain method for detecting virus is laid a good foundation, and has certain application value and realistic meaning.
(3) the present invention provides a kind of Alon mountain virus PCR detection kit, the kit have rapidly and efficiently, operation
Convenient, high specific, high sensitivity, identification is simple, at low cost, do not need expensive instrument, is suitble to the beneficial effects such as on-site test.
Detailed description of the invention
Fig. 1 is the electrophoresis result figure of embodiment 1PCR amplification；Wherein, M indicates DNA Marker DL2000,1,2 and N1 table
Show that the amplification with primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2,3,4 and N2 indicate to use primer ALSV-N5-
The amplification of F1, primer ALSV-N5-F2 and primer ALSV-N5-R1；1 and 3 be same amplification template, and 2 and 4 be same amplification
Template, N1 and N2 indicate negative control (deionized water), and arrow is expressed as PCR product.
Fig. 2 is the electrophoresis result figure of 1 multisample PCR amplification of embodiment；Wherein, M indicates DNA Marker DL2000, N table
Show negative control (deionized water), 1 and 3 be the amplification of primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2,2 Hes
4 be the amplification of primer ALSV-N5-F1, primer ALSV-N5-F2 and primer ALSV-N5-R1；1 and 2 be same amplification mould
Plate, 3 and 4 be same amplification template, and arrow is expressed as PCR product.
Fig. 3 is the electrophoresis result figure of 3PCR of embodiment of the present invention amplification sensitivity；Wherein, M indicates DNA Marker
DL2000,1 indicates that primary template 100,2 indicates 10 times of dilutions 10-1, 3 indicate 100 times of dilutions 10-2, 4 indicate 1000 times of dilutions 10-3, 5 indicate 10000 times of dilutions 10-4, 6 indicate 100000 times of dilutions 10-5。
Fig. 4 is the electrophoresis result figure of 4PCR of embodiment of the present invention amplification clinical sample；Wherein, M indicates DNA Marker
DL2000, N indicate negative control (deionized water), and 1~5 indicates that clinical sample to be measured, arrow are expressed as PCR product.
Fig. 5 is the electrophoresis result figure of 5PCR of embodiment of the present invention amplification clinical sample；Wherein, M indicates DNA Marker
DL2000, N1, N2 indicate negative control (deionized water), and 1~15 indicates that clinical sample to be measured, arrow are expressed as PCR product.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
In embodiment, Alon mountain virus (ALSV) is in bibliography (A New Segmented Virus
Associated with Human Febrile Illness in China(N Engl J Med 2019；380:2116-
2125.) it is disclosed in)；
Russian spring-summer encephalitis virus (TBEV) vaccine strain, japanese encephalitis virus (JEV) vaccine strain are commercially available；
Fever is with thrombocytopenic syndromes viral (SFTSV) in bibliography (The first molecular
evidence of severe fever with thrombocytopenia syndrome virus in ticks in
Jilin, Northeastern China (Tick Borne Dis.2016,7 (6): 1280-1283.)) in disclose；
Embodiment 1
(1) design of primers: drawn according to Alon mountain virus gene sequence (genebank accession number MH158417) design amplification
Object pair: primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2；According to Alon mountain virus gene sequence, (genebank is logged in
Number MH158415) second pair of amplimer pair of design: primer ALSV-N5-F1, primer ALSV-N5-F2 and primer ALSV-N5-
R1, nucleotide sequence are as follows:
ALSV-F1:5 '-GTVTTYGTGCCRGGHCTGAC-3 '；
ALSV-R1:5 '-GTGGABGGRGTTATBAYYCCYTTNG-3 '；
ALSV-R2:5 '-TGGCABGTRTCRAAMACWGCRTC-3 '；
ALSV-N5-F1:5 '-AGRTCHCTVACSKCTGACCCG-3 '；
ALSV-N5-F2:5 '-CCMSARTGGACRGMRGAGGC-3 '；
ALSV-N5-R1:5 '-TCGATGAGAGCACTCACGAATCTG-3 '；
Wherein, V, Y, R, H, B, N, M, W, S, K indicate to annex base code, V=A/G/C；Y=C/T；R=A/G；H=A/
C/T；B=G/C/T；N=A/T/C/G；M=A/C；W=A/T；S=C/G；K=G/T；
(2) extraction of total serum IgE and the synthesis of cDNA:
1. extracting Alon mountain virus (A New Segmented Virus Associated with using TRIzol method
Human Febrile Illness in China(N Engl J Med 2019；380:2116-2125.)) total serum IgE；
2. removal genomic DNA reaction system: 5 × gDNA Eraser Buffer, 2 μ L, gDNA Eraser1 μ L is prepared,
1. 100~1000 μ g of total serum IgE obtained, no RNA enzyme water are supplemented to 10 μ L, 42 DEG C of reaction 2min and are placed on 4 DEG C step；
3. prepare reverse transcription reaction system: step 2. react after 10 μ L, PrimeScript RT Enzyme of reaction solution
I 1 μ L, RT Primer Mix of Mix, 4 μ L, 5 × PrimeScript Buffer 4 μ L, no 1 μ L of RNA enzyme water amount to 20 μ L, mix
It is even；37 DEG C of heating 15min, 85 DEG C of heating 5s, ice bath at least 1min immediately, is put into -20 DEG C and saves backup later.
(3) PCR system is prepared, using cDNA made from step (2) as template, respectively with primer ALSV-F1 and primer ALSV-
R1 and primer ALSV-N5-F1 and primer ALSV-N5-R1 is amplimer, carries out first round PCR amplification, and specific PCR amplification is anti-
Answer system as shown in table 1:
1 first round of table pcr amplification reaction system
| 10×Buffer | 2.5μL； |
| 10mM dNTP | 0.5μL； |
| Primer ALSV-F1 (or primer ALSV-N5-F1) | 1μL； |
| Primer ALSV-R1 (or primer ALSV-N5-R1) | 1μL； |
| cDNA | 2μL； |
| Archaeal dna polymerase (Tag enzyme) | 0.3μL； |
| ddH2O | Complement to 25 μ L； |
First round pcr amplification reaction program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C extend
30s, totally 10 recycle；94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extension 30s, 25 recycle；72 DEG C of extension 10min；
(4) prepare PCR system, using the obtained first round PCR product of step (3) amplification as template, with primer ALSV-F1 with
Primer ALSV-R2 and primer ALSV-N5-F2 and primer ALSV-N5-R1 is amplimer, carries out the second wheel PCR amplification；
Table 2 second takes turns pcr amplification reaction system
| 10×Buffer | 2.5μL； |
| 10mM dNTP | 0.5μL； |
| Primer ALSV-F1 (or primer ALSV-N5-F2) | 1μL； |
| Primer ALSV-R2 (or primer ALSV-N5-R1) | 1μL； |
| First round PCR product | 2μL； |
| Archaeal dna polymerase (Tag enzyme) | 0.3μL； |
| ddH2O | Complement to 25 μ L； |
Second wheel pcr amplification reaction program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C extend
30s, totally 10 recycle；94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extension 30s, 25 recycle；72 DEG C of extension 10min；
(5) PCR reacts amplified production analysis: the second wheel PCR product addition for taking the amplification of 5 μ L steps (4) to obtain respectively contains
In 1 (W/V) % Ago-Gel of nucleic acid dye, the electrophoresis 25min under 130V pressure stabilizing, gel imaging system observation amplification item
Band.224bp signature band is shown on electrophoretogram using primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2 amplification,
218bp feature is shown on electrophoretogram using primer ALSV-N5-F1, primer ALSV-N5-F2 and primer ALSV-N5-R1 amplification
Property band, then determine result for the positive；Such as without any band, then determine result for feminine gender.
As a result as shown in Figure 1, sample No. 1 and No. 2 is existed using primer ALSV-F1, primer ALSV-R1 and primer ALSV-R2
The signature band of Alon mountain virus can be detected at 224bp；Sample No. 3 and No. 4 utilize primer ALSV-N5-F1, primer
ALSV-N5-F2 and primer ALSV-N5-R1 cannot detect signature band at 218bp, wherein No. 1 and No. 3 from same
The serum sample of one patient, No. 2 and No. 4 serum samples for deriving from the same patient.
The amplification efficiency of above-mentioned two groups of primers is further evaluated using multisample, specific method is same as above.
Wherein, primer ALSV-F1, primer ALSV-R1 and the positive findings of primer ALSV-R2 detection have 9 parts, 8 parts negative,
Obtained result and virus purification cultivation results are completely the same, and the positive rate of detection is 100%, and primer ALSV-N5-F1,
The positive findings of primer ALSV-N5-F2 and primer ALSV-N5-R1 detection have 3 parts, and 14 parts negative, the positive rate of detection is
33.33%, partial results are shown in Fig. 2, illustrate that the detection effect of primer pair ALSV-F1, ALSV-R1, ALSV-R2 are drawn better than primer pair
Object ALSV-N5-F1, primer ALSV-N5-F2 and primer ALSV-N5-R1, by primer ALSV-F1, primer ALSV-R1 and primer
ALSV-R2 is the PCR detection primer of invention as this.
2 specific test of embodiment
According to 1 step of embodiment (2) specific steps, Alon mountain virus (ALSV), forest are extracted using TRIzol method respectively
Encephalitis viruses (TBEV) vaccine strain, japanese encephalitis virus (JEV) vaccine strain and fever are with thrombocytopenic syndromes virus
(SFTSV)(The first molecular evidence of severe fever with thrombocytopenia
syndrome virus in ticks in Jilin,Northeastern China(Tick Borne Dis.2016,7(6):
Total serum IgE 1280-1283.)), and reverse transcription, obtain cDNA, using cDNA as template, with primer ALSV-F1, primer ALSV-R1
It is amplimer with ALSV-R2, carries out first round PCR amplification and the second wheel PCR amplification, specific pcr amplification reaction system and PCR
Amplified reaction program is shown in embodiment 1, wherein negative control is deionized water, and positive control is that Alon mountain is viral (ALSV).
It the results are shown in Table 3, only 224bp signature band can be detected in positive control Alon mountain viral (ALSV), remaining is to be checked
Viral sample illustrates that virus PCR detection primer group in Alon mountain provided by the invention has preferable specificity without characteristic bands.
The specific test of 3 Alon mountain virus PCR detection primer of table
| Test number | Virus to be checked | Amplification |
| 1 | ALSV | + |
| 2 | TBEV | — |
| 3 | JEV | — |
| 4 | SFTSV | — |
| 5 | ddH2O | — |
The test of 3 sensitivity of embodiment
Alon mountain virus (ALSV) cDNA for being 100ng/ μ L by concentration made from embodiment 2, does 10 times of dilutions, is followed successively by
100、10-1、10-2、10-3、10-4With 10-5, using primer ALSV-F1, primer ALSV-R1 and ALSV-R2 as amplimer, carry out the
One wheel PCR amplification and the second wheel PCR amplification, specific pcr amplification reaction system and pcr amplification reaction program are shown in embodiment 1,
In, negative control is deionized water, detects the Monitoring lower-cut of PCR primer provided by the present invention.
As a result as shown in figure 3,10-3Purpose band, that is, minimum detectable 100pg/ μ L also can be detected in dilution
CDNA template.
The detection of 4 clinical sample of embodiment
5 parts of doubtful Alon mountain virus infection clinical samples are acquired, all samples all pass through cell culture and carry out virus purification.
Using method described in embodiment 1, PCR detection is carried out to all samples, as a result as shown in figure 4, in 5 parts of clinical samples, Ah
The positive findings of Longshan virus PCR detection have 1 part, 4 parts negative, obtained result and virus purification cultivation results complete one
It causes.
The detection of 5 clinical sample of embodiment
15 parts of doubtful Alon mountain virus infection clinical samples are acquired, all samples all carry out virus point by cell culture
From.Using method described in embodiment 1, PCR detection is carried out to all samples, as a result as shown in figure 5, in 15 parts of clinical samples
In, the positive findings of Alon mountain virus PCR detection have 8 parts, and 7 parts negative, obtained result and virus purification cultivation results are complete
It is complete consistent.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, 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>Foshan Science &. Technology College
<120>a kind of Alon mountain virus PCR detection primer group, kit and application
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-F1
<400> 1
gtvttygtgc crgghctgac 20
<210> 2
<211> 25
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-R1
<220>
<221> misc_feature
<222> (24)..(24)
<223> n is a, c, g, or t
<400> 2
gtggabggrg ttatbayycc yttng 25
<210> 3
<211> 23
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-R2
<400> 3
tggcabgtrt craamacwgc rtc 23
<210> 4
<211> 21
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-N5-F1
<400> 4
agrtchctva cskctgaccc g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-N5-F2
<400> 5
ccmsartgga crgmrgaggc 20
<210> 6
<211> 24
<212> DNA
<213> Artificial
<220>
<223>primer ALSV-N5-R1
<400> 6
tcgatgagag cactcacgaa tctg 24
Claims (9)
1. a kind of Alon mountain virus PCR detection primer group, it is characterised in that include primer ALSV-F1, primer ALSV-R1 and primer
ALSV-R2, nucleotide sequence are as follows:
Primer ALSV-F1:5 '-GTVTTYGTGCCRGGHCTGAC-3 '；
Primer ALSV-R1:5 '-GTGGABGGRGTTATBAYYCCYTTNG-3 '；
Primer ALSV-R2:5 '-TGGCABGTRTCRAAMACWGCRTC-3 '；
Wherein, V, Y, R, H, B, N, M and W indicate degeneracy base code, V=A/G/C；Y=C/T；R=A/G；H=A/C/T；B=
G/C/T；N=A/T/C/G；M=A/C；W=A/T.
2. application of the Alon mountain virus PCR detection primer group described in claim 1 in the viral diagnosis of Alon mountain.
3. a kind of Alon mountain virus PCR detection kit, it is characterised in that include Alon mountain described in claim 1 virus PCR
Detection primer group.
4. Alon mountain virus PCR detection kit according to claim 3, it is characterised in that comprising 10 μM of ALSV-F1,
10 μM of ALSV-R1,10 μM of ALSV-R2,10mM dNTP, 10 × Reaction Buffer, archaeal dna polymerase and deionized water.
5. application of the Alon mountain virus PCR detection kit in the viral diagnosis of Alon mountain described in claim 3 or 4.
6. a kind of method of PCR detection Alon mountain virus, characterized by comprising the steps of:
(1) it extracts the RNA of sample to be tested and reverse transcription is cDNA；
(2) prepare PCR system, using cDNA made from step (1) as template, with above-mentioned Alon mountain virus PCR detection primer group or
Primer ALSV-F1 and primer ALSV-R1 in above-mentioned Alon mountain virus PCR detection kit are amplimer, carry out the first round
PCR amplification；
(3) PCR system is prepared, the first round PCR product obtained using step (2) amplification is template, with above-mentioned Alon mountain virus PCR
Detection primer group or primer ALSV-F1 in above-mentioned Alon mountain virus PCR detection kit and primer ALSV-R2 are that amplification is drawn
Object carries out the second wheel PCR amplification；
(4) the second wheel PCR product for obtaining step (3) amplification carries out agarose gel electrophoresis detection, occurs mesh at 224bp
Band, then be determined as the positive, do not occur purpose band at 224bp, be then determined as feminine gender.
7. the method for PCR detection Alon mountain virus according to claim 6, it is characterised in that:
PCR system described in step (2) includes following component:
8. the method for PCR detection Alon mountain virus according to claim 6, it is characterised in that:
PCR system described in step (3) includes following component:
9. the method for PCR detection Alon mountain virus according to claim 6, it is characterised in that:
The reaction condition of PCR amplification described in step (2) and step (3) are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 50 DEG C
Anneal 30s, 72 DEG C of extension 30s, totally 10 circulations；94 DEG C of denaturation 30s, 48 DEG C of annealing 30s, 72 DEG C of extension 30s, 25 recycle；
72 DEG C of extension 10min.
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| CN113604612A (en) * | 2021-09-03 | 2021-11-05 | 广东方道基因生物科技有限公司 | Alongshan virus loop-mediated isothermal amplification detection primer group, kit containing primer group and application of kit |
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