LU502017B1 - Nested PCR detection primer set and method for identifying African swine fever gene deletion strain - Google Patents

Nested PCR detection primer set and method for identifying African swine fever gene deletion strain Download PDF

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LU502017B1
LU502017B1 LU502017A LU502017A LU502017B1 LU 502017 B1 LU502017 B1 LU 502017B1 LU 502017 A LU502017 A LU 502017A LU 502017 A LU502017 A LU 502017A LU 502017 B1 LU502017 B1 LU 502017B1
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seq
gene deletion
primer
deletion strain
pcr
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LU502017A
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French (fr)
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Pan Zeng
Ziwei Zhang
Yu Wang
Shaoxian Chen
Guoping Liu
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Univ Yangtze
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    • 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
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    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

This invention provides nested PCR detection primer set and method for identifying African swine fever gene deletion strain. According to the invention, a pair of outer primers and a pair of inner primers are designed for pDP71L gene deletion strain, DP96R gene deletion strain, A276R gene deletion strain and MGF360-12L gene deletion strain respectively, and the nucleotide sequences of the eight pairs of primers are shown in SEQ ID NO.1-16. According to the invention, the outer primer and the inner primer are respectively used for PCR amplification, the first amplification strengthens the specificity of PCR, the second amplification strengthens the sensitivity of PCR detection, and two rounds of PCR improve the specificity and sensitivity of PCR, and the sensitivity of the method is as low as one order of magnitude, and even viruses with extremely low load can be detected.

Description

Description LU502017 Nested POR detection primer set and method for identifying African swine fever gene deletion strain Technical field The invention belongs to the technical field of molecular diagnosis, particularly relates to nested PCR detection primer set and method for identifying African swine fever gene deletion strain.
Background African swine fever (ASF) is caused by African swingfevervirus (ASFV) which can cause acute hemorrhagic fever and lead to high morbidity and mortality of pigs.
African swing fever virus belongs to the DNA virus order, the African swing fever virus family, a member of the African swine fever virus Genus. African swine fever (ASF) is a double-stranded linear DNA virus with icosahedral structure, diameter of 175 ~ 215 am, genome length of 170 ~ 190 KB, t has 151 open reading frames, and can encode 150 ~ 200 proteins, and has double-stranded linear DNA virus with envelope.
Ai present, the research of African swing fever vaccine mainly focusses on inactivated vaccine, attenuated vaccine, virus live vector vaccine, nucleic acid vaccine and so on. Inactivated vaccine refers to the use of physical or chemical methods to remove pathogenic activity, loss of infection ability, but still has antigenicity.
Up to now, ASFV inactivated vaccine prepared by many traditional methods cant provide effective immune protection for pigs. Aftenuated vaccines can be divided into Ihres types according to different strains: natural attenuated strains, passage atlenuated strains and recombinant attenuated strains, À large number of experimental results show that there are still problems in biological safely of natural aitenuated straing and passaged attenuated strains. Al present, researchers in China have developed a series of recombinant atienuated vaccines based on the genotypes of the African classical swine fever virus prevalent in China.
Due to insufficient research data on vaccines with cenetic deletion of African swing fever virus, ds specific immune effect still needs further study. In the process of using vaccine, it is necessary to distinguish and identify the wild strain of ASFV from the vaccine strain with gene deletion, so as to correctly identify the infection source of pigs 502017 that show African swing fever symptoms but having done vaccine injection, In recent years, domestic and foreign scholars have established fluorescent antibody test, common PCR diagnosis, real-time fluorescent quantitative PCR and other technologies to detect the gene deletion vaccine of African swine fever virus.
SUMMEN in view of this, the purpose of the present invention is to provide nested PCR detection primer set and method for identifying African swine fever gene deistion strain. in this method, the outer primer and the inner primer are respectively used for two PCR amplifications, so that the specificity and sensitivity of PCR are improved, the sensitivity is as low as 1 order of magnitude, thus, the viruses with extremely low volume can be detected.
in order io achieve the above object, the present invention provides the following technical solution: The invention provides nested PCR detection primer set and method for identifying African swing fever gene deletion strain, aiming at oDP71L gene deletion strain, the nucleotide sequence of the outer primer is shown as SEQ ID NOT and SEQ ID NO 2, and the nucleotide sequence of the inner primer is shown as SEQ ID NO. and SEQ ID NO À With respect to the DP96R gene deletion strain, the nucleotide sequence of the outer primer is shown in SEQ ID NO S and SEG 1D NOS, and the nuciectide sequence of the inner primer is shown in SEQ 1D NO.7 and SEQ ID NOB; With respect to AZ76R gene deletion strain, the nucleotide sequence of the outer primer is shown in SEQ ID NGO and SEQ ID NGO, and the nucleotide sequence of the inner primer is shown in SEQ ID NOT and SEQ ID NO AZ, With respect to the MGF360-12L gene delstion strain, the nucleotide sequence of the outer primer is shown in SEQ ID NO 13 and SEQ ID NO 14, and the nucleotide sequence of the inner primer is shown in SEQ ID NOS and SEG 1D No 16, À nested PCH detection kit for identifying African swine fever gene delstion strains, and the kit comprises the primer set of claim 1.
Freferably, the kit further comprises a PCR amplification reagent.
Preferably, the PCR amplification reagent comprises SU/ u | TaKaRabx Tag, 20MM 502017 107 Ex Tag Buffer, dNTP, outer primer or inner primer, and template.
Frefsrably, the kit further comprises 3 positive reference substance and a negative reference substance.
Preferably, the positive reference substance contains plasmid DNA of African swine fever virus pDF71L or African swing fever virus DPSSR or African swine fever virus AZ276R or African swine fever virus MGF360-12L gene deletion strain, À nested POR detection method for identifying African swine fever gene deistion strains for non-disease diagnosis purposes comprises the following steps: S1, extracting viral nucisic acid from a sample; 52, taking the viral nucleic acid of S1 as a template, and performing the first PCR amplification reaction with the outer primer of claim 1 to obtain an amplification product À 83, taking the amplification product À of 52 as a template, and performing a second PCR amplification reaction with the inner primer of claim 1 to obtain an amplification product B; 34, performing agarose gel electrophoresis analysis on the amplification product B described in 83, and observing the results under a gel imaging system to determing the virus type.
Preferably, the specific band of the second PCR amplification is: the specific band fragment of pDP71L gene deletion strain is 11850; the specific band fragment of DPOGR gene deletion strain is 17450; the specific band fragment of AZ76R gene deletion sirain is 228bp; the specific band fragment of MGF360-12L gene deletion strain is 18/bp.
Preferably, the first PCR amplification reaction conditions of 82 include: pre-denaturing at 88°C for 46 min; denaturing at 88°C for 105, annealing at 55°C for 35s, and stretching at 72°C for 45s, a total of 25 cycles; at last, stretching at 72°C for Amin.
Preferably, the second POR amplification reaction conditions of $3 include: pre-denaturing at 85°C for 5 min, denaturing at 98°C for 10s, annealing at 57°C for 38s, and streiching at 72°C for 45s, a total of 35 cycles; at last, streiching at 72°C for 4mirt Compared with the prior art, the invention has the bensficiai effects: (1) The invention uses the designed outer primer and inner primer to respectively carry out two POR amplifications, the first amplification enhances the specificity of PCR, the second amplification enhances the sensitivity of POR detection, and the probability of nonspecific combination in the second amplification is extremely low, and the sensitivity of this method is as low as Torder of magnitude, so that viruses with extremely low 1044502017 can be detected.
{2} The present invention only produces specific amplification reaction for the DNA of African swine fever virus (ASF V) but does not amplify the nucleic acids of porcine circovirus type 2 (PCY2), porcine parvovirus (PPV), porcine pseudorabies virus (PR), porcine reproductive and respiratory syndrome virus (PRRSWV), the nucleic acids of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (POVS) have no amplification reaction, so they have strong specificity, {3} The present invention provides a new means for clinical detection of African swing fever virus in China, The kit is convenient and practical in clinical application, and it can be widely used in early warning monitoring, clinical monitoring and differential diagnosis of environmental samples, feed raw materials, personnel, food and pig samples.
Brief Description Of The Figures Fig. 1 shows the results of lateral specificity test of nested PCR of ASFVODP/1E gene deletion strain.
Fig. 2 shows the results of nested PCR inner apecificity test of ASFVpDP71L gene deletion strain.
Fig. 3 shows the results of nested POR lateral specificity test of ASFV DPOSR gene deletion strain Fig. 4 shows the results of nested PCR inside specificity test of ASFV DPSGR gene deletion stram, Fig. 5 shows the results of nested POR lateral specificity test of ASFVAZYER gene deletion strain, Fig. 5 shows the results of nested PCR inner specificity test of ASFVAZTER gene deletion sirain, Fig. 7 shows the results of lateral specificity test of nesied PCR of ASFV MGEFSGO-12L gene deletion strain, Fig. 8 shows the results of nesied POR inside specificity test of ASFY MGF360-120 gene delstion strain Wharsin,
in Fig, 1- Fig. 8, 1 ASFV pDP71L gene deletion strain or ASFY DFOBR Sent 502017 deletion strain or ASFVAZTER gene deletion strain or ASFV MGF360-12L gene deistion strain; 2: Forcine circovirus type 2 (POV2) 3: Forcine parvovirus (PPV); 4: Pseudorabies virus {PRY}, 5 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV © Porcine epidemic diarrhea virus (PEDV)Y, 7. Porcine circovirus type 3 {(POV3), = Negative control, 5 Reagent control.
Fig, © shows the sensitivity test results of outerprmers of nested PCR of ASFYpDP74L gene deletion strain, Fig. 10 shows the results of sensitivity test of inner primer of nested PCR of ASFYpDP74L gene deletion strain.
Fig. 11 shows the results of sensitivity test of ouler primers of nesied POR of ASF V DPSSKR gene deletion strain, Fig. 12 shows the results of sensitivity test of inner primer of nested POR of AGFV DRIER gene deletion strain.
Fig. 13 shows the results of sensitivity test of outer primers of nested PCR of AGFVAZTOR gens deletion strain.
Fig. 14 shows the sensitivity test results of the inner primer of nested PCR of ASFVAZTER gene deletion strain.
Fig. 15 shows the results of sensitivity test of outer primers of nested PCR of ASFV MGF350-12E gene deletion strain.
Fig. 16 shows the sensitivity test results of the inner primer of nesisd PCR of ASFV MGF360-12L gene delstion strain.
in Fig. S-Fig. 16, wherein, M is DL2000 DNAMarker, and 1-11: the number of template copies is 1x10°0 1x10% 1x105 1x107, 110% 1x405 4x10* 1x105 4. 13: negative control, 14 reagent control, Fig. 10, 12, 14, 16, 1-13; The first PCR product is used as the corresponding template.
Fig. 17 is à gel siectrophoresis diagram of the detection of interference resistance of the outer primers of nested PCR of ASFV pDP71L gene deletion strain, Fig. 18 is a gel electrophoresis diagram of the interference resistance of the inner primer of nested PCR of ASFV pDP71L gene delstion strain.
Fig. 1818 a gel electrophoresis diagram of the detection of interference resistance 1502017 outer primers of nested POR of ASFV DPYSR gene deletion strairt Fig. 20 is a gel electrophoresis diagram of the anti-interference detection of the inner primer of Nested PCR of AGFV DPSGRE gens deletion strain.
Fig. 21 is a gel electrophoresis diagram of the detection of interference resistance of outer primers of Nested POR of ASFVAZ276R gene deletion strain.
Fig. 22 15 à gel electrophoresis chart of interference resistance detection of the inner primer of Nested PCR of ASFVAZTOR gene deletion strair Fig. 23 is a gel electrophoresis diagram of interference resistance detection of nested POR cuter primers of ASFY MGF360-12L gene deletion strain.
Fig. 24 is a gel siectrophoresis diagram of the interference resistance of the inner primer of Nested PCR of ASFV MGF360-12L gens deletion strain.
Wherein, in Fig. 17- Fig. 24, Mis DL2000 DNAMarker, 1-4: the genome used in the specificity test is mixed with African swine fever pDF71L. DPSOGR, A276R and MGF3650-12E gene deletion strains respectively, 5: negative control, 6: Reagent control.
Fig. 25 is a gel elecirophorssis diagram of a sample detected by using the nested FOF inner primer of ASF V pDF71L gene deletion strain, Fig. 26 is a gel electrophoresis diagram of a sample detecisd by using the nested PCR inner primer of DP96R gene deletion sirain, Fig. 27 is a gel elecirophoresis disgram of a sample detected by using the nested PCR inner primer of AZ76R gene deletion strain.
Fig. 28 is a gel slectrophoresis chart of samples detected by using nested PCR inner primers of MGE380-12L gene delstion strain, Wherein, in Fig 25-Fig 28, M is DL2000 DNAMarker, the numbers 1-50 is samples, and 51 is negative control, 52 is pPD71L fragment control or DFOSK fragment control or A276R fragment control or MGF350-12L fragment control respectively.
Description of the present invention The invention provides a nested POR detection primer set for identifying African classical swing fever gens deletion strains, aiming at pDP71L gene deletion strain, the nucleotide sequence of the cuter primer is shown as SEQ ID NO and SEQ 1D NO 2,
and the nuclectids sequence of the inner primer 1s shown as SEQ ID NO3 and SEQ HY Us02017 MNO À With respect to the DP9SR gene deletion strain, the nucleotide sequence of the outer primer is shown in SEG ID NOS and SEQ ID NG .G, and the nucleotide sequence of the inner primer is shown in SEQ 1D NO.7 and SEQ ID NG 8, With respect to AZTER gene deletion strain, the nucieotide sequence of the outer primer is shown in SEQ ID NC.9 and SEQ ID NOC, and the nucleotide sequence of the inner primer is shown in SEQ ID NOT and SEQ ID NO 12; With respect to the MGF380-12L gene deletion strain, the nucleotide sequence of the outer primer is shown in SEQ ID NO 13 and SEQ ID NO 14 and the nucleotides sequence of the inner primer is shown in SEQ ID NOS and SEQ id No 18.
in this invention, the outer primer for pDP71L gene deletion strain is ASFY-DDP71L-F17/R1, and the inner primer is ASFV-pDP7IL-F2/R2, The outer primer for the DPSOR gene delstion strain is ASFV-DPOSR-FÜRT1, and the inner primer is asfy-doS0r-F2/R2; The outer primer for A276R gene delstion strain is ASFY-AZISR-F1/RT, and the inner primer is ASFV-AZT7ER-FIR2Z, The outer primer for the MGF360-12L gene delstion strain is ASFV-MGF360-12L-F1/R1, and the inner primer is ASFV-MGF360-12L-F2/R2.
in the present invention, the kit further comprises a PCR amplification reagent The PCR amplification reaction system in this invention includes TaKaRa Ex Tac(SU/uL}, 10xEx Tag Buffer(20md), dNTP, outer primer or inner primer, and template. The amount of TaKaRa Ex Tag{&Uiul) is preferably 0.125uk, the amount of 10xEX TagBuffer(ZOmML is preferably 2.5ul, the amount of dNTP is preferably Zul, and the amount of outer primer or inner primer is preferably Tull, preferably, the template is 2 pul: the outer primer is ASFY-pDP7TIL-FI/RT or ASFV-DPSOR-F URI or ASFV-AZTER-FURI or ASFV-MGFSSU-1T2L-FÜRT, and the inner primer is ASFY-pDP71L-F2/R2 or ASFV-DPOOR-F2/R2 or ABFV-AZYVBR-FZIRZ of ASFY-MGFIG0-12L-F2/R2.
in the present invention, the kit also includes a positive reference substance and a negative reference substance. The positive reference subsiance in the invention contains plasmid DNA of African swine fever virus pDF71L or African swine fever virus DRIER or African swine fever virus AZ76R or African swing fever virus MGF380-12L gene delstion strain, and the negative reference substance is preferably deionized 502017 water.
The invention also provides a nested POR detection method for identifying African swing fever gens deletion strains for non-disease diagnosis purposes, which comprises the following siens: 31, extracting viral nucleic acid from a sample; Se, taking the viral nucleic acid of 51 as a template, and performing the first PCR amplification reaction with the outer primer of claim 1 to obtain an amplification product A; $3, taking the amplification product À of 82 as a template, and performing a second FOR amplification reaction with the inner primer of claim 1 10 oblain an amplification product B; 54, performing agarose gel slectrophoresis analysis on the amplification product 8 described in 83, and observing the results under a gel imaging system to determine the virus type.
in the present invention, the specific band of the first PCR amplification is. the specific hand fragment of pDP71L gens delstion strain is 206bo, The specific band fragment of DPSSR gene deletion strain is 266bp; The specific band fragment of A276R gene deletion strain is 450bp, the specific band fragment of MGFS360-12L gene delstion strain is 688bp. The specific band of the second PCR amplification is: the specific band fragment of pDP71L gene deletion strain is 118bp, the specific band fragment of DPSOR gene deletion strain is 17460; the specific band fragment of AZ7BR gens delstion strain is 228bp, the specific band fragment of MGFSG0-12L gene deletion strain is 187bp.
in the present invention, the first PCR amplification reaction system comprises: D 125uL TaKaRa Ex Tao(SU/uL} 2.501 10xEx TagBuffer(ZOmM} Zui dNTP, Tul outer primer pair, Zul template, and deionized water to 25uL; the first PCR amplification template is viral nucieic acid extracted from a sample, The reaction system of the second PCR amplification includes: 0.125ul TaKaRa Ex Taq(6U/uL) 2.5uL 10xEx Tag Buffer(Z20mid), ZULONTP, Tub inner primer pair, 2uL template, supplemented with delonized water to 25ul, and the second amplification template is the product of the first PCR ampiification,
in the present invention, the first PCR amplification reaction conditions include: pre-denaturation at 98°C for 4-6 min; denaturing at 98°C for 10s, annealing at 55°C for 25s, and stretching at 72°C for 45s, a total of 35 cycles, at last, stretcning at 72°C for Amin, the reaction conditions of the second PCR amplification include: pre-denaturing at 95°C for bir denaturing at 95°C for 10s, annealing at 57°C for 35s, and stretching at 72°C for 45s, a total of 35 cycles; At last, stretching at 72°C for 4min. in the present invention, the criteria for determining viruses are shown in Table 1. Moemaifrens VDPTIL DPOGR AZTER MOP 360-121 * FREE > Detection Frama PENSE Fragma Fragman _ + = + + + Wid Minus + - + + + poPTiL Let etion Shan + + » + + DD Deletion strains + & + ~ + AFTER Deletkın sain + + + + = SH ABEL TEL, Pete siraine + - = + + SER ATTHR odds gene daletlan strains © _ + . . plP7IL, AI7ER gun © + + | pDPTIL, MOF360-J0L ; Cote gene delation slrains ; ; ; DPAGE, ATER Double sana deletion strains + ; + DPS6R, MUFI00-12L {ratée gene deleting shan N _ N ATER.
MOFIG EN Double gens deletion strains N N PORT, DPSSR, A276R ; ; Triple gone dele stradya pDPTIL, TIPOSR, + > + > MT int 1 24, Triple gene deletion strains The technical solutions provided by the present invention will be explained in detail with reference to the following Embodiments, but they should not be understood as imiting the scope of protection of the present invention.
Embodiment 1 Frimer design in this embodiment, the genes of pDP71L, DPSSR, A276R and MGF 360-121 gen 502017 deletion strains of African swing fever virus (ASF) published in GenBank database of NCBI (National Center for Biological Information) are highly conserved and have specific regions. The specific outer and inner primers of pDP/4L, DPOSR, AZVBR and MGF3650-12L gene deletion strains of ASFY were designed. À large number of experiments show that different primers have certain influence on the effect and sensitivity of PCR amplification. Therefore, in this embodiment, two sets of primers were designed for the above four basic fragments, thus providing a nested POR primer set for identifying ASFV gene deletion strains, as shown in the following table 2 for details. Table 2 Amplification primers of Tour ASFY deletion strains Primer Sequence Sequence Number Fragment Size/BP ASPV-pDPTILAF ATCGOGGOOGCOGUUNCG SEQIDNOI ASEY-POPTIL-RI TOCASITAGOTIPPITTG SEG ID NO ASFYSpDPTIL-F2 OTATOOGAAGCCGADGACS SEO IY NOI 18g ARFY-HEIPTIL-RI OC AÏGATH ACCOTGGATAG SEO FI Wed
ABFVADPOER FI ASAMTATAASITAG SEC ID NOS 268hy
ASFUHIPOGR HI TTAAŸTATTOTTOYGGA SH ID NOLS ASFV-DPSER FI GAAATOATCTGROGTGGA SEQ ID NOT Pid ASFY-DFAGR 22 TPIGTERRIACTGOTCATTT SEQ I 808 ASFV-AITSE-F! TATYCCTUCTUCTTTGUC HEQIDNGS SShp ASFY-AITER-RI TCCOONTUTOTOGATIOT KEN FD NON ASFYSATIOR-FI AGTAGATOATUTUUCGANTA SEQ BD NON Bly ASPY-AITER-H2 ACGHAGUATTITAATACT SEQ HIND ASPV-MOPIM-ITEFL GAGTTAGATGOCAAGNAG BEQID NOY
EN ASFVMOPISH- ILL GATUTATGATTTOAGERST SECHD NOS ASFW-MOTIOIZE-P2 TOAATACOTOOCGOTTAA SECHEENOLES 1$7bp ASFY-MGFID-IILRI AGCATUGCCTOATTUATG SEDIDNOIS Embodiment 2 FOR detection method
1.1 primer in Embodiment 1
1.2 Sample DNA extraction
Extraction of viral DNA from tissue samples [ LU502017 À Taking fresh or just thawed tissue to remove blood and connective tissue, and cutting it into small pieces on an ice bath.
B Moving the tissue fragments Into a precocled homogenizer, adding homogenizing buffer (homogenizing buffer: 0.25mmolil sucrose, ZommobL Tris-HOHpH
7.5}, 25mmol/ NaCl and 25mmo/MgaChb} at 10mg, and mixing uniformly to prapare homogeneous SIUMY ©. Moving the homogenate into Ep tube, centrifuging at 5000r/ min for 10min, and discarding the supernatant; D Adding 10mmolLPBS{pHT 4) io suspend the precipitate, adding tml per 01g of original tissue, centrifuging at 5000r/min for 10min, and discarding the supernatant; E. Adding lysate {the lysate consists of S0mmoyL Tris-HCKpHB.3), mmol. EDTA,
0.5% tween-20 and 200 ug/Mmi protesse K (selected from TIANGEN, RT403-02}) to suspend and precipitate, adding 1m to every D 19 of original tissue, reacting at 55°C for 30-60min, and then inactivating protease K at 95°C for 10min; Centrifuging at &. 1000 mun for Smin, taking the supematant as DNA template, and storing the exiracted DNA at -20°C for later use or immediately use à for POR amplification.
it, Extraction of virus DNA from body fluid: À. Adding an equal amount of lysis buffer A (lysis buffer A S0OmmobL Tris-HCT (pH
7.4) 150mmoi Nall into 750ul whole blood, mixing well, centrifuging at 1 2000r/rmin for 30s, and discard the supernatant B. Suspending the pracipitate with 1.5m lysis buffer À, centrifuging once more, and discarding the supamatant.
©. Dissolving and precipitating with 117UL lysis buffer B (lysis buffer 8: BümmolLTris-HCT {ph 8.3), trmmol/LEDTA, 0.5% tween-20), at 70°C for Smin, when it was cooled to 56 C, adding Sul protease K and kesping the temperature for 1h to digest the cells, inactivating protease K at 95°C for 10min.
D. Adding 30ul of Smo/L KCL solution and dissolving in ice for Smin, E. Centrifuging at 12000/min for Smin, discarding supernatant as DNA template, and storing the extracted DNA ai -20°C for later use or immediately use it for FOR amplification,
1.3 Positive plasmid LU502017 The full length of genes of pDP71L, DPOGR, A2756R and MGF360-12L gene deletion strains of ASFYV published in GenBank database were sent to Shanghal Shenggong Synthetic Gene, and the positive plasmids were named pUC57-pDP71L pUC57-DPGGR, pUC57-A276R and pUC57-MGF3S0-12E,
1.4 Sensitivity test of nested PCR reaction The four plasmids pUGS/7-pDP71L, pUC57-DPOSR, pUCS/-A276R and pUCS/-MGF350-12L obtained in 1.3 are diluted, and the measured concentrations are 3 14x10" copice/ul, 3.08x 107% copice/ul and 2.88x 10" copice/uL, and 288x107 copice/ul, and the four plasmids were diluted to 110"? copice/uL as templates.
Using POR instrument, different primer concentration combinations are screened by matrix method, in order io obtain the primer concentration and reaction conditions for nested PCR.
Nested PCR is performed according to the following steps: S1, extracting viral nucleic acid from a sample: S52, taking the nucleic acid extracted in step S1 as a template, and performing the first PCR amplification reaction with the outer primer described in claim 1 to obtain an amplification product; the amplification system include Ù 125uL TaKaRa Ex Tag{SU/ 1},
2.50 10xEx TagBuffer(20mM}, Zul dNTP, TuUl outer primer pair, Zul template, and supplementing deionized water to 25uL; PCR reaction conditions are. pre-denaturing at 98°C for 4-6 min; denaturing at 88°C for 10s, annealing at 55°C for 35s, and stretching at 72°C for 45s, a total of 35 cycles, At last, strefching at 72°C for Amin.
53, taking that amplified product in step SZ as a template, and performing a second PCR amplification reaction with the inner primer of claim 1 to obtain an amplified product B; The amplification reaction system include: 0.125ul TaKaRa Ex Tag(GU/uL} 2.5uL 10xEx TagBuffer(ZOmM), 2uldNTP, Tul inner primer pair, Zul template, supplementing with deionized water io 25uL; PCH reaction conditions are: pre-densturation at 98°C for Smin; denaturing at 98°C for 10s, annealing at 57°C for 35s, and stretching at 72°C for 48s, a total of 35 cycles, Al last, streiching at 72°C for Amin.
34, performing agarose gel electrophoresis analysis on the PCR ampliicetion 502017 product in $3, and observing the result under a gel imaging system to determine the virus type.
Electrophoretic identification of PCR products: weighing 2g agarose in a SDOmL conical flask, adding 100mL of x TAE electrophoresis buffer, melling it in a microwave oven, and then adding 10ul of staining solution (selected from Qingke Bio TS-GelRed nucleic acid gel dye, TSJ002) to mix well Putting the comb in the electrophoresis tank mold, pouring the agarose gel, taking the comb out and put if in the electrophoresis tank after # is completely solidified, spotting 5 ul of FCR amplification products in the agarose gel hole, and carrying oul electrophoresis in 1x TAE electrophoresis buffer at 10V voltage, and observing the results by gel imaging system, Embodiment 3 Specificity test According to the nested PCR detection method established in 1.4 in Embodiment 2, 4 ASFV PDP 71] DPOGR, AZ76R and MGFSG0-12L gene delstion strains, 2 Porcine circovirus type 2 (PCVS), 3. Porcine parvovirus (PPV), à Pseudorabies Virus (PRY), 5: porcine reproductive and respiratory syndrome virus (PRRSY) ©: porcine epidemic diarrhea virus (PEDV) 7. porcine circovirus type 3 (PCV3) 8 negative control, © reagent control, The results are shown in Fig 1-5, it can be seen from Fig. 1-2 that the lane corresponding to 1: AGFVDDP71L gene deletion strain can ses a clear band at the position of the corresponding fragment size, 2 porcine circovirus type 2 (POV2) 3 Porcine parvovirus (PPV) 4 Pseudorabies virus {FRY}, 5: Porcine Reproductive and Respiratory Syndrome Virus (PRESVX 6 Porcine gpidemic diarrhea virus (PEDWV) 7: Porcine circovirus type 3 (PCVS) 8 negative control,
9. Compared with the reagent, the corresponding lanes didn't show any bands, which indicated that the identification method had good specificity.
it can be seen from Fig. 3-4 that the lane corresponding to 1: ASFV DP96R gene deletion strain can see a clear band at the position of the corresponding fragment size, while 2: porcine circovirus type 2 virus (PCV2) 3 Porcine parvovirus (PPV), 4 Pseudorabies virus (PRVY 5 Porcine Reproductive and Respiratory Syndrome Virus {(PRRSYY 6 Porcine epidemic diarrhea virus (PEDV) 7: Porcine circovirus type 3
{FCVS) 8 Negative control © Compared with the reagent, the corresponding BOGS 502017 didnt show any bands, which indicates that the identification method had good specificity.
it can be seen from Fig. 5-6 that the lane corresponding to 1: ASFVAZTER gene deletion strain can see a clear band at the position of the corresponding fragment size, and © Forcing circovirus type 2 (PCVZ) 3; Porcine parvovirus (PPV), 4 Pseudorabies virus (PRY), 5: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV © Porcine epidemic diarrnea virus (PEDV) 7: Porcine circovirus type 3 (POV3y 8 Negative control 9: Compared with the reagent, the corresponding lanes didn't show any bands, which indicated that the identification method had good specificity.
it can be seen from Fig.7-8 that a clear band can be seen at the position of the corresponding fragment size in the lane corresponding to 1: ASFV MGF2360-12L gene deletion strain, and 2; Porcing circovirus type 2 (PCYZY 3 Porcine parvovirus (PPV), 4. Pseudorabies virus (PRVY 5 Porcine Reproductive and Respiratory Syndrome Virus {(PRRSYY 6 Porcine epidemic diarrhea virus (PEDV) 7: Porcine circovirus type 3 {FCVS3) 8: Negative control (deionized water) © Compared with the reagent, the corresponding lanes didnt show any bands, which indicated that the identification method had good specificity.
Embodiment 4 Sensitivity test The positive templates pUGS&/-pDP71L, pUCE7-DPIBR, pUCS7-A276R and pUC57-MGF360-12L were diluted 10 times to the 11% gradient, The PCR detection results of the outer primer of African swine fever pDP71L gene deletion strain are shown in Fig 8. Clear bands can be seen in lanes 1 to 7, indicating that the PCR sensitivity of the outer primer is 1x 10% copicel/ul; The PCR detection results of the inner primers are shown in Fig. 10, and clear bands can be seen in lanes 1 to 11, indicating that the POR sensitivity of the inner primers is Toopice/ul.
The PCR detection results of the outer primer of African swine fever DPOCK gene deletion strain are shown in Fig 11. Clear bands can be seen in lanes 1 to 9 indicaling that the PCR sensitivity of the outer primer is 1x 102 copice/ul, The FOR detection results of the inner primers are shown in Fig. 12, and clear bands can be seen in lanss 502017 to 14, indicating thai the PUR sensitivity of the inner primers is 1copice/uL.
The results of POR detection of the outer primer of A276R gene delstion strain of African swine fever are shown in Fig 13. Clear bands can be seen in lanes 1 to 6, indicating that the PCR sensitivity of the outer primer is 1x 10% copice/uL: The PCR detection results of the inner primers are shown in Fig 14, and dear bands can be seen in lanes 1 to 11, indicating that the PCR sensitivity of the inner primers is Tcopice/uL.
The POR detection results of the auter primer of African swing fever MGF360-12L gene deletion strain are shown in Fig 15, Clear bands can be seen in lanes 1 to 5, indicating that the PCR sensitivity of the outer primer is 1x 10% copice/uL: The PCR detection results of the inner primers are shown in Fig 16, and clear bands can be seen in lanes 1 to 11, indicating that the PCR sensitivity of the inner primers is 1copice/uL.
Embodiment & interference test The genome used in the specificity test was mixed with the deletion gene strains of African classical swine fever pFD71L, DP96R, AZVER and MOF360-12L in the same amount, The outer primer and the inner primer are used for PCH amplification, respectively, in order to check the anti-interference of the primers and whether the target band could be amplified from the mixed genome template. Results as shown in Fig. 17-24, wherein, the clear bands can be seen in lanes 1 to 4 indicating that the primers have strong anti-interference ability.
Embodiment 6 Conformance Test From January to April, 2021, 50 ASFV positive samples were collecied from a town in Hubei province, including 20 samples from vegelable market environment, 15 samples from pig farms and 15 samples from vehicles. The samples were numbered from 1 to 50, and the DNA was extracted by the nucleic acid extraction procedure described in 1.2 of Embodiment 2.
The external primers of pPD71L, DPOSKR, A276R and MGFS60-12L gens deletion strains of African swine fever are used for the first amplification, and the FOR amplification reaction system and amplification reaction conditions are as described 1502017 S2 in Embodiment 2.
The inner primers of oPD71L, DPOGKR, AZ7GR and MGF360-12L gene delstion strains of African swine fever are used for the second amplification. The PCR amplification reaction system and amplification reaction conditions are as described in S3 in Embodiment 2.
The second POR product is detected by electrophoresis using 2% agarose gel as described in 54 in Embodiment 2. As shown in Fig, 25-28, No.8 amplified pPD71L and MGF360-12L, No. 12 amplified A276HK, No.17 amplified A276R and MGFS00-12L, No 24 amplified MGFSG0-12L, and No 47 amplified DPSSR, According to Fig, 25-28, among 50 samples, one sample is deleted with pPD71L and MGF360-12L, one sample is deleted with MGF360-12L, one sample is deleted with A276R and MGF3G0-12L, and two samples are deleted with DP96R and A276R genes respectively.
The above are only the preferred embodiments of the present invention, and it should be pointed out that for those of ordinary skill in the technical field, without departing from the principle of the present invention, several improvements and embellishments can be made, and these improvernents and embellishments should also Ge regarded as the protection scope of the present invention.
Sequence List <110> Yangtze University <120> Nested POR detection primer set and method for identifying African swings fever gene deistion strain <130> PT2020 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence
<400> 1 LU502017 atgggsggec ggcggcg 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 tccagtaget ttttttg 17 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 gtatgggaag ccgacgaca 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 gcaggagcac cgtggatag 19 <210> 5 <211> 14 <212> DNA <213> Artificial Sequence <400> 5 aaaagagaaa ccag 14 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <400> 6 ttaattattc ttctgga 17 LU502017 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 gaaatcatct gtcgtgga 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tttgtctgea ctggtcattt 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 tattcctget cctetgge 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <400> 10 tccgegtetg tggattgt 18 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 agtagatgat gtgccgagta 20
<210> 12 LU502017 <211> 18 <212> DNA <213> Artificial Sequence <400> 12 acggagcagt tgaatacc 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <400> 13 gagttaggtg ccaaggag 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <400> 14 gatctatgat ttcagggt 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <400> 15 tgaatacgtg gcggttaa 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <400> 16 agcatggcet gattgatg 18

Claims (10)

Claims
1. A nested PCR detection primer set for identifying African swing fever gene deletion strain is characterized in that, aiming at pDP/71L gene deletion strain, the nucleotide sequence of the cuter primer is shown as SEQ ID NO and SEQ ID NO 2, and the nucleotids sequence of the inner primer is shown as SEQ ID NO3 and SEQ ID NG 4; with respect to the DP96R gens deletion strain, the nuclectide sequence of the outer primer is shown in SEG ID NOS and SEQ ID NG .G, and the nucleotide sequence of the inner primer is shown in SEQ ID NO.7 and SEQ ID NO 8; with respect to AZ76R gene deletion strain, the nuciectide sequence of the outer primer is shown in SEQ ID NO.9 and SEQ ID NOC, and the nucleotide sequence of the inner primer is shown in SEQ ID NOT and SEQ ID NO 12; with respect to the MGF36D0-12L gene deletion strain, the nuciectide sequence of the outer primer is shown in SEQ ID NO 13 and SEG ID NO 14 and the nucleotides sequence of the inner primer is shown in SEQ ID NOS and SEQ id No 16,
2. A nested PCR detection kit for identifying African swing fever gene deletion strains is characterized in that the kit comprises the primer set of claim 1.
3. The kit, according to claim 2, is characterized in that the kit further comprises a PCR amplification reagent.
4, The kit, according to claim 3, is characterized in that the PCR amplification reagent comprises SU/ uv L TaKaRakx Tag, 20mM 10XEx Tag Buffer, dNTP, outer primer or inner primer, and template,
5. The kit, according to claim 2, is characterized in that the kit further comprises a positive reference subsiance and a negative reference substance.
6. The kit, according to claim 5, is characterized in that the positive reference substance contains plasmid DNA of African swine fever virus pDP71L or African swine fever virus DPSOR or African swing fever virus AZ7ER or African swine fever virus MGEFSGO-12L gene deletion strain,
7. A nested POR detection method for identifying African swine fever gene deletion strains for non-disease diagnosis purposes IS characterized by comprising the following steps: S1, extracting viral nucleic acid from a sample; 82, taking the viral nucleic acid of
S1 as a template, and performing the first PCR amplification reaction with the outer LU502017 primer of claim 1 to obtain an amplification product À S3, taking the amplification product À of S2 as a template, and performing a second POR amplification reaction with the inner primer of claim 1 to obtain an amplification product B; 54, performing agarose gel electrophoresis analysis on the amplification product B described in 83, and observing the results under a gel imaging system to determing the virus type, & The detection method according to claim 7 is characterized in that the specific band of the second PCR amplification is: the specific band fragment of oDP71L gene deletion strain is 118bp, the specific band fragment of DP9SR gene deletion strain is 174650; the specific band fragment of A27GK gene deletion strain is 228bp; the specific band fragment of MGF360-12L gene deistion strain is 187bp.
9. The detection method, according to claim 7, is characterized in that the first POR amplification reaction conditions of 52 include: pre-denaturing at 88°C for 4-6 mir, denaturing at 88°C for 10s, annealing at 55°C for 35s, and siretching at 72°C for 455, a total of 35 cycles, at last, stretching at 72°C for 4min.
10. The detection method, according to claim 7, is characterized in that the second FOR amplification reaction conditions of S3 include. pre-denaturing at 95°C for 5 mim, denaturing at 88°C for 10s, annealing at 57°C for 358, and stretching at 72°C for 45s, a total of 35 cycles, at last, stretching at 72°C for 4min.
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