LU504681B1 - A Universal Nested RT-PCR Detection Method for Porcine Deltacoronavirus - Google Patents
A Universal Nested RT-PCR Detection Method for Porcine Deltacoronavirus Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
Abstract
The invention provides a nested RT- PCR primer, detection method and kit for detection of porcine deltacoronavirus. The nest RT- PCR primers are two pairs of primers designed according to highly conserved specific sequences for detecting porcine deltacoronavirus. The detection method comprises the following steps: Extracting sample RNA; Using the obtained sample RNA as a template, the primers are used for RT- PCR reaction; The reaction products are analyzed by agarose gel electrophoresis. The kit comprises: primer, amplification reagent, positive control and negative control. The technical scheme provided by the invention has strong specificity, high sensitivity, good anti-interference performance and strong reliability, overcomes the problems of low sensitivity and poor specificity of common detection methods, and has lower cost, short detection period and strong practicability compared with existing detection methods such as nucleic acid hybridization and gene chip.
Description
DESCRIPTION LU504681
A Universal Nested RT-PCR Detection Method for Porcine Deltacoronavirus
The invention relates to the technical field of molecular diagnosis, in particular to a universal nested RT-PCR detection method for porcine deltacoronavirus.
Porcine deltacoronavirus (PDCoV) is a member of 6-coronavirus genus of Coronaviridae, and it is a worldwide epidemic porcine enterovirus discovered in recent years. Its clinical manifestations are acute diarrhea, vomiting and dehydration. The virus is first reported in Hong
Kong in 2012. After 2014, the disease is reported in the United States, South Korea and other countries one after another, and then it broke out and spread around the world, which had a certain impact on the healthy development of the pig industry.
Porcine deltacoronavirus is a member of Deltacoronavirus, belonging to Coronaviridae, and its genome composition and arrangement are similar to other coronaviruses. Its genome includes at least seven ORFs (open reading frames), which encode four structural proteins (nucleoprotein
N, small envelope protein E, fibrillar protein S and membrane glycoprotein M), two non-structural proteins (NS6 and NS7 proteins) and two multimeric proteins (la and 1b proteins) respectively. Coronavirus is the longest in the gene sequence length of known RNA viruses, but the sequence size of porcine deltacoronavirus is about 25.4kb, which is the smallest in genome among all coronaviruses.
The clinical symptoms of PDCoV are similar to those of other porcine enterocoronaviruses.
Therefore, the detection of porcine deltacoronavirus is mainly laboratory detection at present.
Pathogen detection techniques mainly include ordinary RT-PCR, fluorescence quantitative
RT-PCR, LAMP and so on. Antibody detection is the main serological detection method.
However, due to the low sensitivity of ordinary RT-PCR, this patent establishes a nested
RT-PCR method for detecting PDCoV on the basis of ordinary RT-PCR to improve the sensitivity and specificity of detection.
Nested RT-PCR is a variant polymerase chain reaction, which uses two pairs of PCR/S04681 primers to amplify the target fragment. After twice PCR amplification, the sensitivity of common
PCR can be greatly improved, and the probability of nonspecific binding of secondary PCR primers is extremely low. Sequence analysis showed that the amino acids of N protein of epidemic strains are highly conserved and can be used as the target antigen for clinical diagnosis.
In view of this, the invention provides nested RT-PCR primers, detection method and kit for detecting porcine deltacoronavirus, and establishes a universal nested RT-PCR detection method for porcine deltacoronavirus, which has the characteristics of strong specificity and high sensitivity, and is suitable for any laboratory and grass-roots prevention and control units, veterinary stations, large and medium-sized farms and the like, and has important practical significance.
The first aspect of the present invention provides a nested RT-PCR primer for detecting porcine deltacoronavirus The primer designs two pairs of specific primers according to the base sequence in detecting porcine deltacoronavirus N gene, a pair of outer primers: PDCoV-O-F and
PDCoV-O-R, and a pair of inner primers: PDCoV-I-F and PDCoV-I-R, specifically:
PDCoV-O-F: 5'-CAGGTCCCAGAGGAAATCTTA-3'(SEQ ID NO.1);
PDCoV-0-R: 5'-TTTGGTAGGTGGCTCATAGGT-3'(SEQ ID NO 2);
PDCoV-I-F: S-GAAGACCTCAGGAGCGTGGAA-3'(SEQ ID NO 3);
PDCoV-I-R: 5S'-TGAGTAGGAGAAGGTAAGGGTGA-3'(SEQ ID NO.4).
The second aspect of that invention provide a nested RT-PCR detection method for detecting porcine deltacoronavirus, which comprise the following steps:
S1. Extracting RNA of the sample to be detected by using a Trizol RNA extraction solution;
S2. The first round of PCR amplification: using the RNA extracted from S1 as a template, performing the first round of PCR amplification by using the outer primer of claim 1, and performing agarose gel electrophoresis analysis on the PCR amplification products, and bands with the size of 665bp are amplified, it is proved to be positive, otherwise it is negative;
S3. The second round of PCR amplification: using the negative PCR amplification product of the first round as a template, performing the second round of PCR amplification by using the inner primer of claim 1, and performing agarose gel electrophoresis analysis on the PCR/S04681 amplification product, and if bands with the size of 353bp are amplified, it is proved that there is porcine coronavirus in the sample.
Further, in S2, the PCR reaction system is: 2x1 Step Buffer 12.5ul, PrimeScript 1 Step
Enzyme Mix lul, 20umol/L PDCoV-O-F and 20umol/L PDCoV-O-R 1ul each, RNA template ul, add RNase Free dH 2 O to 25ul.
Further, in S2, the reaction conditions of PCR are: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 45s, repeat 32 cycles and extension at 72°C for 6min.
Further, in S3, the PCR reaction system is: Mix 12.5ul, 20umol/L PDCoV-I-F and 20umol/L of PDCoV-I-R 1ul each, template 1pul, and add RNase Free dH 2 O to 25ul.
Further, in S3, the reaction conditions of PCR are: pre-denaturation at 94°C for 2min, denaturation at 98°C for 30s, annealing at 57°C for 30s, extension at 72°C for 45s, 32 cycles and extension at 72°C for 6min.
The third aspect of the present invention provides a kit for detecting porcine deltacoronavirus, which comprises the two pairs of specific primers described in the first aspect of the present invention, as well as an amplification reagent, a positive control and a negative control, wherein the positive control is a recombinant plasmid containing a 665bp gene fragment of porcine deltacoronavirus, and the negative control is RNase-free water.
The nested RT-PCR detection primer provided by the invention has the beneficial effects that the nested RT-PCR detection primer is four specific primers designed for the base sequence in the N gene of the porcine deltacoronavirus, and has specificity for the porcine deltacoronavirus. The lowest copy number of the detection method provided by the invention for detecting the existing epidemic strains can reach one order of magnitude, and the lowest copy number is 1.4, which is 2-3 orders of magnitude higher than that of the common PCR detection method. Using Trizol RNA extract solution to extract RNA from the sample to be tested can not only destroy the cell structure quickly, but also ensure the integrity of RNA and improve the reliability of the test results. Moreover, this method is suitable for the serum, diarrhea, spleen, lung and other samples of pigs to be detected, so this detection method is suitable for any laboratory, grass-roots prevention and control units, veterinary stations, large and medium-sized farms and so on. To sum up, the method provided by the invention has the characteristics 5504681 strong specificity, high sensitivity, strong practicability and the like, and overcomes the problems of low sensitivity, poor specificity and the like of common PCR detection, and has low cost and short detection period.
Fig. 1 is the gel electrophoresis diagram of the specific detection of outer primers of nested
RT-PCR, where M is DL2000DNAMarker; 1 is a recombinant positive plasmid; 2-7 are porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), Escherichia coli and
Staphylococcus aureus; 8 is a negative control (RNase-free water).
Fig. 2 is the gel electrophoresis diagram of the specific detection of inner primers of nested
RT-PCR, where M is DL2000DNA Marker; 1 is a recombinant positive plasmid; 2-7 are porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), Escherichia coli and
Staphylococcus aureus; 8 is a negative control (RNase-free water).
Fig. 3 is the gel electrophoresis diagram of the sensitivity detection of nested RT-PCR outer primers, where M is DL2000DNA Marker; The number of template copies in 1-9 is 1.4x10®, 1.4x107, 1.4x106, 1.4x10°, 1.4x10%, 1.4x10°, 1.4x10?, 1.4x10' and 1.4.
Fig. 4 1s the gel electrophoresis diagram of the sensitivity detection of nested RT-PCR inner primers, and the products of the first PCR are used as the corresponding templates for 1-9 respectively; The number of template copies in 1-9 is 1.4x10$, 1.4x107, 1.4x10° 1.4x10°, 1.4x10% 1.4x10°, 1.4x10% 14x10! and 1.4.
Fig. 5 is the gel electrophoresis diagram of nested RT-PCR primer interference detection, in which M is DL2000DNA Marker, 1 is the mixed genome amplified by outer primers, and 2, 3 and 4 are negative controls (RNase-free water).
Fig. 6 is the gel electrophoresis diagram of nested RT-PCR primer interference detection, in which M is DL2000DNA Marker, 1 is the mixed genome amplified by inner primers, and 2, 3 and 4 are negative controls (RNase-free water).
DETAILED DESCRIPTION OF THE INVENTION LUS04681
The present invention will be further described with embodiments, but it is not to limit the scope of the present invention, but only for illustration.
Embodiment 1: Establishment of nested RT-PCR detection method for porcine deltacoronavirus (1) Design of nested RT-PCR primers for porcine deltacoronavirus
Two pairs of specific primers are designed according to the base sequence in the N gene of porcine deltacoronavirus, and the sequences of the two pairs of primers are as follows:
PDCoV-O-F: 5'-CAGGTCCCAGAGGAAATCTTA-3'(SEQ ID NO.1);
PDCoV-0-R: 5'-TTTGGTAGGTGGCTCATAGGT-3'(SEQ ID NO 2);
PDCoV-I-F: S-GAAGACCTCAGGAGCGTGGAA-3'(SEQ ID NO 3);
PDCoV-I-R: 5S'-TGAGTAGGAGAAGGTAAGGGTGA-3'(SEQ ID NO.4). (2) Sampling and RNA extraction
A: sample collection: collect sample serum, centrifuge at 3000r/min for 20min, take 200uL of Trizol RNA extract to extract RNA, and measure the concentration for later use.
B: tissue sample treatment: the tissues and organs of the pig samples to be detected, such as liver and lung, are taken, cut and then extracted with the Trizol RNA extract, and the concentration is determined for later use. (3) PCR amplification and electrophoresis detection
A: The first PCR amplification is performed with outer detection primers, namely,
PDCoV-O-F and PDCoV-O-R. The PCR reaction system is as follows: 2x1Step Buffer 12.5ul,
PrimeScript 1Step Enzyme Mix 1ul, 20umol/L PDCoV-O-F and 20jumol/L PDCoV-O-R 1ul each, RNA template 1ul, and add RNase Free dH 2 O to 25ul; The PCR reaction procedure is as follows: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 45s, repeat 32 cycles, extension at 72°C for 6min, electrophoresis detection of PCR products with 1% agarose gel, and operate the second amplification if there is no target band,
B: The template of the first amplification product is amplified for the second time with inner detection primers PDCoV-I-F and PDCoV-I-R. The optimum amplification conditions of inner primers are as follows: Mix 12.5ul, 20umol/L PDCoV-I-F and 20jumol/L PDCoV-I-R 1ul each, template 1ul, add RNase Free dH 2 O to 25ul; The procedure of PCR reaction is as follows! °04681 pre-denaturation at 94°C for 2min, denaturation at 98°C for 30s, annealing at 57°C for 30s, extension at 72°C for 45s, repeat 32 cycles and extension at 72°C for 6min. The PCR products are detected by electrophoresis with 1% agarose gel. (4) Result analysis
According to the results of nested RT-PCR amplification, it is judged that after the first
PCR amplification, the target band is detected by electrophoresis and reported as positive, and the sample carries a high amount of virus; After the second PCR amplification, the target band detected by electrophoresis is also judged to contain porcine deltacoronavirus, but the virus carrying amount of the sample is relatively low. If there is no target band after two rounds of
PCR, it is reported that the sample does not contain porcine deltacoronavirus
Embodiment 2: Construction of recombinant positive plasmid pMD18-T-N (1) Cloning of N gene
The genomic RNA of porcine deltacoronavirus (PEDV) is extracted, and the partial conserved sequence of N gene is amplified by nested RT-PCR outer primers PDCoV-O-F and
PDCoV-O-R. The PCR reaction system (25ul) is: 2x1Step Buffer 12.5ul, PrimeScript 1Step
Enzyme Mix lul, 20umol/L PDCoV-O-F and 20umol/L PDCoV-O-R 1ul each, RNA template
Tul, add RNase Free dH 2 O to 25ul; The procedure of PCR reaction is: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 45s, repeat 32 cycles, extension at 72°C for 6min. The PCR product is identified by electrophoresis in 1% agarose gel. As shown in Figure 1, a specific DNA band of 665bp is obtained, which is similar to the target DNA. (2) Construction of positive plasmid pMD18-T-N.
The above PCR products are recovered by the gel recovery kit of TaKaRa company, and then connected with the pMD18-T cloning vector of TaKaRa company. The connection system is: pMD18-T vector 1ul, PCR recovered products 3ul, Solution I Sul, ddH 2 0 1ul; After being connected at 16°C for 30min, the competent cells of Escherichia coli DH5a are transformed and coated on Amp-resistant LB medium. Cultured at 37°C overnight, the correct recombinant is screened out by resistance.
(3) Identification of pMD18-T-N positive recombinant. LU504681
The selected positive clones are identified by colony PCR with primers PRV-O-F and
PRV-O-R, and the correct recombinant is named pMD18-T-N. (4) Extraction of recombinant plasmid pMD18-T-N.
After the correct recombinant 1s identified by sequencing and enriched with LB broth, the plasmid 1s extracted by MiniBEST Plasmid Purification Kit, a high-purity plasmid extraction kit from TaKaRa Company, and the concentration is determined, and the copy number is calculated.
Embodiment 3: Characteristic detection of nested RT-PCR primers (1) Specificity test: The genomes of porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), Escherichia coli and Staphylococcus aureus are extracted as templates, the specificity of two pairs of primers used in nested RT-PCR, namely, PDCoV-O-F&PDCoV-O-R and PDCoV-I-F&PDCoV-I-R is tested, and the results show that the two pairs of primers had good specificity and high amplification efficiency. (2) Sensitivity test: The positive plasmid obtained in Embodiment 2 is diluted by 10 times to single-digit copy number, and the plasmids with different dilutions are selected as template primers for the first PCR amplification, and the PCR products are detected with 1% agarose gel.
The results are shown in Figure 3. The electrophoresis results of the first PCR amplification show that the number of gene copies detected by the outer primers is 10°, and the amplification specificity is good. The amplified product is used as the template of corresponding gradient, and is amplified by PCR for the second time with primers PDCoV-I-F and PDCoV-I-R, and the amplified product is detected with 1% agarose gel. The results are shown in Figure 4, the lowest order of magnitude of gene copy number that can be detected after two rounds of nested RT-PCR amplification. (3) Interference test: Mix the genome used in the specificity test with diarrhea genome in equal amount, and use primers PDCoV-O-F, PDCoV-O-R and PDCoV-I-F, PDCoV-I-R to perform PCR amplification respectively to test the anti-interference of the primers and whether the target band can be amplified from the mixed genome template. The results are shown in
Figures 5 and 6, both pairs of primers can amplify specific bands from the mixed template, which shows that the primers have good anti-interference performance.
(4) Coincidence rate test: In order to verify the sensitivity of nested primers to the detecticht 204681 of porcine deltacoronavirus in various regions, the genomes of HKU15-44, HKU15-155, CH-A,
HB-BD and other strains are extracted, and the number of copies is calculated after measuring the concentration, which is diluted to single-digit copies by gradient. After two rounds of nested
RT-PCR amplification, all types of genomes can detect a gene copy number of one order of magnitude, proving the feasibility of the method of the present invention.
Embodiment 4: Nested RT-PCR reliability test 1. Clinical sample collection: In May 2018, 42 blood samples of piglets are collected from a large-scale pig farm in Henan Province, centrifuged at 3000r/min for 20min, and the serum samples are collected, and RNA is extracted with Trizol RNA extract. 2. PCR amplification and electrophoresis detection: Carry out two rounds of PCR amplification according to step (3) of Embodiment 1. The amplified products are detected by electrophoresis, and 31 of 42 serum samples are positive. The sequencing results showed that all the 31 positive products are porcine deltacoronavirus genes, which proved that the detection results of this method are reliable.
For those skilled in the art, various other corresponding changes and deformations can be made according to the technical scheme and ideas described above, and all these changes and deformations should be within the protection scope of the claims of the present invention.
Sequence table <110> Yangtze University <120> A universal nested RT-PCR detection method for porcine deltacoronavirus. <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211>21 <212> DNA <213> Artificial Sequence <400> 1 caggtcccag aggaaatett a 21 <210> 2
<211>21 LU504681 <212> DNA <213> Artificial Sequence <400> 2 tttggtaggt gectcatagg t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 gaagacctca ggagcgtgga a 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 tgagtaggag aaggtaaggg tga 23
Claims (8)
1. A nested RT-PCR specific primer for porcine deltacoronavirus, characterized by designing two pairs of specific primers according to highly conserved specific sequences for detecting porcine deltacoronavirus, wherein the two pairs of primers are a pair of outer primers, PDCoV-O-F and PDCoV-O-R, and a pair of inner primers, PDCoV-I-F and PDCoV-I-R, specifically: PDCoV-O-F: S'-CAGGTCCCAGAGGAAATCTTA-3'(SEQ ID NO 1); PDCoV-0-R: 5'-TTTGGTAGGTGGCTCATAGGT-3'(SEQ ID NO 2); PDCoV-I-F: S-GAAGACCTCAGGAGCGTGGAA-3'(SEQ ID NO 3); PDCoV-I-R: 5S'-TGAGTAGGAGAAGGTAAGGGTGA-3'(SEQ ID NO.4).
2. A nested RT-PCR detection method for porcine deltacoronavirus, comprising the following steps:
sl. extracting RNA of the sample to be detected by using a Trizol RNA extraction solution;
s2. the first round of PCR amplification: using the RNA extracted from S1 as a template, performing the first round of PCR amplification by using the outer primer of claim 1, and performing agarose gel electrophoresis analysis on the PCR amplification products, and bands with the size of 665bp are amplified, it is proved to be positive, otherwise it is negative;
s3. the second round of PCR amplification: using the negative PCR amplification product of the first round as a template, performing the second round of PCR amplification by using the inner primer of claim 1, and performing agarose gel electrophoresis analysis on the PCR amplification product, and if bands with the size of 353bp are amplified, it is proved that there is porcine coronavirus in the sample.
3. A nested RT-PCR detection method for porcine deltacoronavirus according to claim 2, characterized in that in S2, the PCR reaction system is: 2x1 Step Buffer 12.5ul, PrimeScript 1 Step Enzyme Mix 1ul, 20umol/L PDCoV-O-F and 20umol/L PDCoV-O-R 1ul each, RNA template 1ul, add RNase Free dH 2 O to 25ul.
4. A nested RT-PCR detection method for porcine deltacoronavirus according to claim 2, characterized in that in S2, the reaction conditions of PCR are: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 2min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 45s, repeat 32 cycles and extension at 72°C for 6min.
5. A nested RT-PCR detection method for porcine deltacoronavirus according to claim bU504681 characterized in that in S3, the PCR reaction system is: Mix 12.5ul, 20pumol/L PDCoV-I-F and 20umol/L of PDCoV-I-R 1ul each, template 1pul, and add RNase Free dH 2 O to 25ul.
6. A nested RT-PCR detection method for porcine deltacoronavirus according to claim 2, characterized in that in S3, the reaction conditions of PCR are: pre-denaturation at 94°C for 2min, denaturation at 98°C for 30s, annealing at 57°C for 30s, extension at 72°C for 45s, 32 cycles and extension at 72°C for 6min.
7. Application of the specific primer according to claim 1 in preparation of nested RT-PCR kit for identifying porcine deltacoronavirus.
8. The nested RT-PCR kit for identifying porcine deltacoronavirus according to claim 7, characterized by further comprising: amplification reagent, positive control and negative control, wherein the positive control is a recombinant plasmid containing a 665bp gene fragment of porcine deltacoronavirus, and the negative control is RNase-free water.
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