Real-time PCR assay for detection of African swine fever virus
Group standard T/CVMA 5—2018
Released by the Chinese Veterinary Medical Association
preface
This standard is drafted in accordance with the rules given in GB/T 1.1-2009.
This standard is proposed and centralized by the Chinese Veterinary Medical Association.
The drafting unit of this standard: China Animal Disease Prevention and Control Center, China Institute of Veterinary Drug Control, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Beijing Customs.
The main drafters of this standard: Ni Jianqiang, Wang Chuanbin, Wang Qin, Qiu Huaji, Yin Hong, Yang Lin, Xin Shengpeng, Liu Yanhua, Liu Yang, Song Xiaohui, Zhao Qizu, Luo Yuzi, Liu Zhijie, Zhang Qianyi, Qiao Caixia, Xia Yingju, Yang Jifei, Xu Lu, Gu Xiaoxue, Kang Wenhua, Li Shuo, Bi Yiming.
1. Scope
This standard specifies the technical requirements for reagents, instruments and consumables, operation steps, result judgment, laboratory biosafety and other technical requirements for the real-time fluorescence PCR detection method of African swine fever virus.
This standard is applicable to the detection of African swine fever virus nucleic acid in pig spleen, lymph nodes, blood and other tissues and blood meal.
2. Normative reference documents
The following documents are essential for the application of this document. Where a reference is dated, only the dated version applies to this document. For undated references, the version (including all change orders) applies to this document.
GB 19489 Laboratories – General requirements for biosafety
NY/T 541 Technical specification for collection, storage and transportation of veterinary diagnostic samples
3. Reagents
3.1 DNA Extraction Reagents
See Appendix A for the preparation of DNA extraction reagents, or select a commercially available viral DNA extraction kit and refer to the instructions for DNA extraction.
3.2 2 × PCR buffer
2 × PCR buffers are prepared in Appendix A.
3.3 Primer probes
3.3.1 Use primers and probes for the VP72 gene of African swine fever virus (see Appendix B for nucleotide sequences):
Upstream primer ASF-CADC-rPCRF: 5'-1528-ATAGAGATACAGCTCTTCCAG-1548-3'
Downstream primer ASF-CADC-rPCR: 5'-1660-GTATGTAAGAGCTGCAGAAC-1679-3'
Fluorescent probe ASF-CADC-Probe: 5'-1638-FAM-TATCGATAAGATTGAT-MGB-1653-3'
3.3.2 Primers and probes provided by the World Organisation for Animal Health (OIE) in Chapter 2.8.1 of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals can be used, and the detection procedures and judgment criteria specified in the manual can be used:
Upstream primer ASF-OIE-rPCRF: 5'-1627-CTGCTCATGGTATCAATCTTATCGA-1651-3'
Downstream primer ASF-OIE-rPCR:
5'-1857-GATACCACAAGATCRGCCGT-1876-3'
Fluorescent probe ASF-OIE-Probe:
5'-1761-FAM-CCACGGGAGGAATACCAACCCAGTG-TAMRA-1785-3'
3.3.3 The use of other primers and probes approved by the national agricultural administrative department shall be adjusted accordingly to the detection procedures and judgment standards.
3.4 Negative and positive controls
The preparation methods for the negative and positive controls are shown in Appendix C.
3.5 Other reagents
Disinfectant solution, 5 U/μLTaq DNA polymerase, sterile nuclease-free water, 0.01mol/L PBS (pH 7.2).
See Appendix A for the preparation of disinfectant solution, and Appendix A for the preparation of 0.01mol/L PBS.
4. Instruments and consumables
Analytical balance (inductance 0.1 mg), high-speed benchtop refrigerated centrifuge (centrifugation speed not less than 12 000 r/min), ice box, real-time fluorescence PCR instrument and supporting reaction tubes (plates), tissue grinder, -20 °C freezer, adjustable pipette (2 μL, 20 μL, 200 μL, 1 000 μL), 1.5 mL centrifuge tube (nuclease-free).
5. Steps
5.1 Sample collection and transportation
Sample collection and transportation in accordance with the provisions of NY/T541, the collection of pig spleen, lymph nodes, blood and other tissue materials or blood meal for testing, samples should be transported to the laboratory as soon as possible under refrigerated conditions to avoid repeated freezing and thawing. Personal biosafety protective equipment should be worn during sampling, and on-site disinfection and waste disposal should be implemented.
5.2 Sample Handling
Samples should be processed in a secondary biosafety cabinet prior to testing. 0.1g~0.2g of tissue or blood meal was taken and 1mL of 0.01mol/L PBS (pH7.2) was added after grinding and crushing, and the supernatant was obtained by centrifugation at 12 000 r/min for 2 min. The samples treated above were inactivated at 60°C for 30 min.
5.3 Sample preservation
The collected or processed samples should be stored at 2°C~8°C for no more than 24 h, and if long-term storage is required, they should be stored in a -70°C freezer, but repeated freeze-thaw (no more than 3 freeze-thaw times) should be avoided.
5.4 Viral DNA extraction
5.4.1 DNA extraction should be performed in the sample preparation area using the following methods, and if other equivalent viral DNA extraction reagents are used, follow the reagent instructions.
5.4.2 The sum of the number of samples to be tested, the positive control and the negative control is expressed by n, and n sterilized 1.5 mL centrifuge tubes are taken and numbered one by one.
5.4.3 Add 200 μL of DNA extract 1 to each tube, then add 200 μL of each of the sample to be tested, the negative control and the positive control respectively, replace 1 sample with 1 pipette, and shake and mix well on the mixer for 5s. Centrifugation at 4°C~25°C at 13 000 r/min for 10 min. See Appendix A for DNA Extraction Solution 1.
5.4.4 Aspirate the supernatant as much as possible, discard it, do not touch the pellet with the pipette tip, add 10 μL of DNA extract2, shake on the mixer and mix well for 5 s. Centrifugation at 4°C~25°C at 2 000 r/min for 10s. See Appendix A for DNA extraction solution 2.
5.4.5 Dry bath or boiling water bath at 100°C for 10 min.
5.4.6 Add 90 μL of DNase-free sterilized deionized water, centrifuge at 13 000 r/min for 10 min, the supernatant is the extracted DNA, and store at -20°C for later use. See Appendix A for DNase-free sterilized deionized water.
5.5.1 5.5.2~5.5.4 in the reaction mixture preparation area, sample preparation area and detection area, respectively.
5.5 Real-time PCR operation
5.5.2 20 μL of real-time fluorescent PCR reaction should be used for each detection reaction. According to the n value set in 5.4.2, prepare the reaction solution according to Appendix D, mix well and aliquot 20 μL per PCR reaction tube. Transfer the PCR reaction tube to the sample preparation area.
5.5.3 Add 5 μL of the DNA solution extracted in 5.4 to the reaction tube of 5.5.2 above to bring the total volume of each tube to 25 μL, and record the sample number corresponding to the reaction tube. After the tube cap is tightly closed, centrifuge instantaneously.
5.5.4 Put the reaction tubes after 5.5.3 injection into the real-time fluorescence PCR detector, and record the order of the reaction tubes. 5-carboxyfluorescein (FAM) was selected as the reporter group and small groove binder (MGB) was selected as the quencher, and the reaction parameters were set as follows: pre-denaturation 95°C/3 min, 95°C/15 s, 52°C/10 s, 60°C/35 s, 45 cycles, and fluorescence was collected at 60°C annealing extension of each cycle. At the end of the test, the results were determined based on the collected Ct values and fluorescence curves.
6. Result judgment
6.1 Result analysis condition setting
The threshold setting principle of real-time fluorescence PCR detection: the threshold line exceeds the higher point of the negative control amplification curve, and intersects at the inflection point when the positive control amplification curve enters the exponential growth period, or is adjusted according to the instrument noise. The number of cycles that the fluorescence signal in each sample tube goes through when it reaches the set domain value is the Ct value.
6.2 Description and Determination of Results
When the Ct value of the positive control was ≤ 28.0 and a typical amplification curve appeared, and the negative control had no Ct value and no amplification curve, the experiment was established, and the example is referred to Appendix E. When the sample with a typical amplification curve and the Ct value ≤ 38.0, it is judged to be positive for ASFV nucleic acid, and the sample with no Ct value is judged to be negative for ASFV nucleic acid, and for the sample with a Ct value of >38.0 and a typical amplification curve appears, it should be retested, and if the above results still appear in the retest, it will be judged to be positive, otherwise it will be judged negative.
7. Laboratory biosafety requirements
7.1 The experimental operation of African swine fever infectious samples of this method should be carried out in the (animal) biosafety level 3 laboratory, and the laboratory biosafety management requirements are described in GB 19489. The State agricultural administrative departments provide otherwise, in accordance with its provisions.
7.2 Used laboratory equipment and liquid waste should be soaked in disinfectant first, and then discarded after high temperature and high pressure treatment. Solid wastes such as remaining samples should be sealed and packaged in a biosafety cabinet, removed after surface disinfection, and then discarded after high-temperature and high-pressure treatment.
1. Appendix A (Normative Appendix) Preparation of reagents
A.1 DNA Extraction Solution 1
PEG8000 crystals 20.74g, NaCL17.53g, and deionized water to 100 mL.
A.2 DNA Extract 2
1mol/L Tris. Hcl 2 mL, 2 mol/L KCL5 mL, 0.5 mol/L EDTA 0.5 mL, NP-40 1 mL, plus deionized water to 100 mL. That is, KCL 14.912g, Tris base 12.114g, 1.2068 mL concentrated HCl, EDTA 14.612g, NaOH 6g, plus deionized water to set the volume to 100 mL.
A.3 2× PCR buffer
Sterilized deionized water 70 mL
Tris(hydroxymethyl)aminomethane (Tris) 0.79 g
Potassium chloride 1.865 g
Traton X-100 0.5 mL
dATP (100mmol/L) 2.5 mL
dTTP (100mmol/L) 2.5 mL
dGTP (100mmol/L) 2.5 mL
dCTP (100mmol/L) 2.5 mL
Magnesium chloride hexahydrate 0.61 g
Hydrochloric acid: Adjust the pH to 9.0
Sterilized deionized water Add to 100 mL
A.4 Disinfectant
Other disinfection reagents such as 8‰ sodium hydroxide or 3‰ formalin or 3% o-phenylphenol or iodine compounds can be used.
A.5 Formulation of phosphate buffered saline (PBS).
A.5.1 A liquid
0.2mol/L sodium dihydrogen phosphate aqueous solution: NaH2PO4· H2O 27.6 g, dissolved in distilled water, to a volume of 1 000 mL.
A.5.2 Liquid B
0.2mol/L dibasic sodium phosphate aqueous solution: Na2HPO4·7H2O 53.6 g (or Na2HPO4·12H2O 71.6 g or Na2HPO4·2H2O 35.6 g), dissolved in distilled water, and the volume was reduced to 1 000 mL.
A.5.3 Preparation of 0.01 mol/L, pH 7.2 phosphate buffered saline (PBS).
14 mL of solution A and 36 mL of solution B, 8.5 g of NaCl were added, and the volume was reduced to 1 000 mL with distilled water. After filtration and sterilization, it is dispensed under sterile conditions.
A.6 DNase-free sterilized deionized water
DNase-free sterilized deionized water is deionized water treated with 1% DEPC and the resistance should be greater than 18.2 mΩ.
2. Appendix B (Informational Appendix) African Swine Fever Virus VP72 Gene Reference Sequence
1ATGGCATCAG GAGGAGCTTT TTGTCTTATT GCTAACGATG GGAAGGCCGA CAAGATTATA
61TTGGCCCAAG ACTTGCTGAA TAGCAGGATC TCTAACATTA AAAATGTGAA CAAAAGTTAT
121GGGAAACCCG ATCCCGAACC CACTTTGAGT CAAATCGAAG AAACACATTT GGTGCATTTT
181AATGCGCATT TTAAGCCTTA TGTTCCAGTA GGGTTTGAAT ACAATAAAGT ACGCCCGCAT
241ACGGGTACCC CCACCTTGGG AAACAAGCTT ACCTTTGGTA TTCCCCAGTA CGGAGACTTT
301TTCCATGATA TGGTGGGCCA TCATATATTG GGTGCATGTC ATTCATCCTG GCAGGATGCT
361CCGATTCAGG GCACGTCCCA GATGGGGGCC CATGGGCAGC TTCAAACGTT TCCTCGCAAC
421GGATATGACT GGGACAACCA AACACCCTTA GAGGGCGCCG TTTACACGCT TGTAGATCCT
481TTTGGAAGAC CCATTGTACC CGGCACAAAG AATGCGTACC GAAACTTGGT TTACTACTGC
541GAATACCCCG GAGAACGACT TTATGAAAAC GTAAGATTCG ATGTAAATGG AAATTCCCTA
601GACGAATATA GTTCGGATGT CACAACGCTT GTGCGCAAAT TTTGCATCCC AGGGGATAAA
661ATGACTGGAT ATAAGCACTT GGTTGGCCAG GAGGTATCGG TGGAGGGAAC CAGTGGCCCT
721CTCCTATGCA ACATTCATGA TTTGCACAAG CCGCACCAAA GCAAACCTAT TCTTACCGAT
781GAAAATGATA CGCAGCGAAC GTGTAGCCAT ACCAACCCGA AATTTCTTTC ACAGCATTTT
841CCCGAGAACT CTCACAATAT CCAAACAGCA GGTAAACAAG ATATTACTCC TATCACGGAC
901GCAACGTATC TGGACATAAG ACGTAATGTT CATTACAGCT GTAATGGACC TCAAACCCCT
961AAATACTATC AGCCCCCTCT TGCGCTCTGG ATTAAGTTGC GCTTTTGGTT TAATGAGAAC
1021GTGAACCTTG CTATTCCCTC AGTATCCATT CCCTTCGGCG AGCGCTTTAT CACCATAAAG
1081CTTGCATCGC AAAAGGATTT GGTGAATGAA TTTCCTGGAC TTTTTGTACG CCAGTCACGT
1141TTTATAGCTG GACGCCCCAG TAGACGCAAT ATACGCTTTA AACCATGGTT TATCCCAGGA
1201GTCATTAATG AAATCTCGCT CACGAATAAT GAACTTTACA TCAATAACCT GTTTGTAACC
1261CCTGAAATAC ACAACCTTTT TGTAAAACGC GTTCGCTTTT CGCTGATACG TGTCCATAAA
1321ACGCAGGTGA CCCACACCAA CAATAACCAC CACGATGAAA AACTAATGTC TGCTCTTAAA
1381TGGCCCATTG AATATATGTT TATAGGATTA AAACCTACCT GGAACATCTC CGATCAAAAT
1441CCTCATCAAC ACCGAGATTG GCACAAGTTC GGACATGTTG TTAACGCCAT TATGCAGCCC
1501ACTCACCACG CAGAGATAAG CTTTCAGGAT AGAGATACAG CTCTTCCAGA CGCATGTTCA
1561TCTATATCTG ATATTAGCCC CGTTACGTAT CCGATCACAT TACCTATTAT TAAAAACATT
1621TCCGTAACTG CTCATGGTAT CAATCTTATC GATAAATTTC CATCAAAGTT CTGCAGCTCT
1681TACATACCCT TCCACTACGG AGGCAATGCG ATTAAAACCC CCGATGATCC GGGTGCGATG
1741ATGATTACCT TTGCTTTGAA GCCACGGGAG GAATACCAAC CCAGTGGTCA TATTAACGTA
1801TCCAGAGCAA GAGAATTTTA TATTAGTTGG GACACGGATT ACGTGGGGTC TATCACTACG
1861GCTGATCTTG TGGTATCGGC ATCTGCT
3. Appendix C (Normative Appendix) African Swine Fever Virus Nucleic Acid Positive and Negative Control
C.1 Positive control
Preparation method of positive control: artificial synthesis of African swine fever virus VP72 gene fragment, the sequence is shown in Appendix B, the VP72 gene was ligated to the pMD20-T vector to make the positive plasmid pMD20-T-VP72, and the plasmid was diluted to a concentration of 10 000copies/mL using the tissue slurry of African swine fever virus negative pigs, and stored at -20 °C for later use.
C.2 Negative control
The negative control was tissue slurry from ASFV-negative pigs.
4. Appendix D (Normative Appendix) Real-Time Fluorescence PCR Reaction Solution Formulation
The recipe for the real-time fluorescent PCR reaction is shown in Table D.1.
Table D.1 Real-time fluorescence PCR reaction recipes
5. Appendix E (Informational Appendix) African Swine Fever Virus Real-time Fluorescence PCR Amplification Example Reference
Figure D.1 shows an example of real-time fluorescence PCR amplification of African swine fever virus.
Figure E.1 Schematic diagram of typical amplification curves of African swine fever virus nucleic acid by real-time fluorescence PCR
