AU2021104086A4 - Detection reagent for fluorescent duplex pcr for classical swine fever virus and african swine fever virus, kit, and detection method - Google Patents
Detection reagent for fluorescent duplex pcr for classical swine fever virus and african swine fever virus, kit, and detection method Download PDFInfo
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- 241000710777 Classical swine fever virus Species 0.000 title claims abstract description 66
- 241000701386 African swine fever virus Species 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 30
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- 101150057545 p72 gene Proteins 0.000 claims abstract description 9
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- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims 2
- 108020004414 DNA Proteins 0.000 claims 2
- 239000012807 PCR reagent Substances 0.000 claims 2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
Disclosed are a detection reagent for fluorescent duplex PCR for classical swine fever virus
(CSFV) and African swine fever virus (ASFV), a kit, and a detection method, and the present
disclosure belongs to the field of animal pathogen detection. In the present disclosure, primers and
probes that are both capable of covering all virulent strains and suitable for dual detection are designed
and screened based on a 5'-untranslated region (5'-UTR) gene of CSFV and a p72 gene of ASFV,
respectively, including two specific primer pairs and two specific probes. The present disclosure
further depicts a kit containing the primers and the probes and a PCR detection method using the
primers and the probes. Through the primer design and the adjustment of all PCR components, and
on the premise that both sensitivity and specificity are not decreased, the present disclosure realizes
an objective of simultaneously detecting and distinguishing between CSFV and ASFV by a one-off
analysis; the present disclosure not only reduces the workload and the cost of detection, but also
substantially saves the detection time, thus winning valuable time to control epidemic diseases.
Description
[01] The present disclosure belongs to the field of animal pathogen detection, and particularly relates to a detection reagent for fluorescent duplex PCR for classical swine fever virus and African swine fever virus, a kit, and a detection method.
[02] Classical swine fever virus (CSFV) belongs to the genus Pestivirusof the family Flaviviridae. The virus is a positive-sense single-stranded RNA virus and is a pathogen of swine fever; the virus endangers hogs and wild pigs, but does not attack other animals.
[03] At present, fluorescent RT-PCR or fluorescent PCR methods for detecting CSFV and African swine fever virus (ASFV) alone and patents thereof have been reported. However, a detection reagent for fluorescent duplex PCR for both CSFV and ASFV, a preparation method and use thereof are rarely reported.
[04] A first objective of the present disclosure is to provide primers and probes for fluorescent PCR for detecting CSFV and ASFV.
[05] A second objective of the present disclosure is to provide a fluorescent duplex PCR kit for detecting and identifying CSFV and ASFV.
[06] A third objective of the present disclosure is to provide a fluorescent duplex PCR method for identifying CSFV and ASFV.
[07] Compared with fluorescent monoplex PCR, through the primer design and the adjustment of all PCR components, and on the premise that both sensitivity and specificity are not decreased, the present disclosure realizes an objective of simultaneously detecting and distinguishing between CSFV and ASFV by an one-off analysis; the present disclosure not only reduces the workload and the cost of detection, but also substantially saves the detection time, and thus winning valuable time to control epidemic diseases.
[08] Compared with the prior arts, the present disclosure has the following beneficial effects:
[09] (1) Excellent coverage and universality: The primers and the probes in the present disclosure are designed for the most conservative genes of the CSFV and the ASFV, cover all sequences of the viruses existed in Genebank, and do not lead to missed detection. In addition, amplification conditions formed by the primers and the probes in the present disclosure are the same as those of several other important porcine infectious diseases, so that experiments may be carried out simultaneously on the same platform together with other epidemic diseases (porcine infectious diseases). Thus, excellent universality is a research and development objective of medical diagnosis at present, aiming to make the diagnosis faster and more convenient; this is a main innovation point of the present disclosure.
[10] (2) Multiplicity and high throughput: The fluorescent duplex PCR may simultaneously realize rapid identification and detection of the CSFV and the ASFV in one reaction, and the detection may be completed within 65 min, saving the detection time and costs. This may satisfy the requirement of mass detection and be conducive to timely diagnosis of epidemic diseases, especially mixed infection disease, thus winning valuable time to control epidemic diseases.
[11] (3) High sensitivity: The fluorescent duplex PCR detection method has comparable sensitivity to the corresponding fluorescent monoplex PCR detection method, thus overcoming the shortcoming of a decrease in sensitivity of commercial fluorescent multiplex PCR kits in China at present.
[12] (4) Strong specificity: Possible interferences among pathogens have been taken fully into account when designing the primers and the probes. It has been demonstrated that the fluorescent duplex PCR has no cross reaction with other common important infectious diseases pathogens in pigs, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), foot-mouth disease virus (FMDV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), porcine epidemic diarrhea virus (PEDV), and porcine transmissible gastroenteritis virus (TGEV).
[13] (5) The cycle threshold of the present disclosure is increased from 40 cycles commonly used in kits made in China at present to 45 cycles commonly used in imported kits, and the determination criterion for results is increased from 35 cycles commonly used to 38 cycles, resulting in further improvement of integral levels of kits; this is another innovation point of the present disclosure.
[14] Fig. 1 illustrates gene-specific amplification curves of CSFV and ASFV in the present disclosure. In the figure, 1 illustrates amplification curves of a positive control of the CSFV; 2 illustrates amplification curves of a positive control of the ASFV; 3 to 11 are amplification curves of a negative control, PRRSV, PRV, FMDV, PCV2, PPV, JEV, PEDV, and porcine TGEV.
[15] Fig. 2 illustrates amplification curves of CSFV gene in the present disclosure.
[16] Fig. 3 illustrates a standard curve of CSFV gene in the present disclosure.
[17] Fig. 4 illustrates amplification curves of ASFV gene in the present disclosure.
[18] Fig. 5 illustrates a standard curve of ASFV gene in the present disclosure.
[19] Fig. 6 illustrates a comparison of amplification curves of fluorescent monoplex PCR versus duplex PCR for CSFV gene in the present disclosure. In thefigure, 1 illustrates amplification curves of fluorescent monoplex PCR for CSFV; 2 illustrates amplification curves of the CSFV in fluorescent duplex PCR for CSFV and ASFV.
[20] Fig. 7 illustrates a comparison of amplification curves of fluorescent monoplex PCR versus duplex PCR for ASFV gene in the present disclosure. In thefigure, 1 illustrates amplification curves of fluorescent monoplex PCR for ASFV; 2 illustrates amplification curves of the ASFV in fluorescent duplex PCR for CSFV and ASFV.
[21] Example 1:
[22] A fluorescent duplex PCR method for detecting and identifying CSFV and ASFV was established.
[23] The first step was to design and synthesize primers and probes of the CSFV and the ASFV:
[24] Based on the NCBI's sequence alignment analysis, primers and probes that are both capable of covering all virulent strains and suitable for dual detection were designed and screened according to a 5'-untranslated region (5'-UTR) gene of CSFV and a p72 gene of ASFV, respectively, including two specific primer pairs and two specific probes, amplified target fragments were 94 bp and 249 bp in length, respectively, and the sequences of the primers and the probes were as follows:
[25] CSFV:
[26] forward primer: F: 5'-TGCCCAYAGTAGGACTAG-3' (SEQ ID NO:1);
[27] reverse primer: R: 5'-CTACTGACGACTGTCCTG-3' (SEQ ID NO:2);
[28] probe: P: 5'-TGGCGAGCTCCCTGGGTGGTCTA-3'(SEQ ID NO:3);
[29] FAM and TAMRA were labeled at the 5'-end and 3'-end of the probe, respectively.
[30] ASFV:
[31] forward primer: F: 5'-GATACCACAAGATCTGCCGT-3'(SEQ ID NO:4);
[32] reverse primer: R: 5'-CTGCTCATGGTATCAATCTTATCG-3'(SEQ ID NO:5);
[33] probe: P: 5'-CCACGGGAGGAATACCAACCCAGTG-3'(SEQ ID NO:6);
[34] VIC and BHQ were labeled at the 5'-end and 3'-end of the probe, respectively.
[35] The second step was to prepare positive controls of the CSFV and the ASFV:
[36] (1) At NCBI, the sequence of the 5'-UTR gene of the CSFV was aligned, and a fragment with conserved sequence and containing the foregoing primers was selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The synthesized fragment was ligated to a cloning vector and transformed into genetically engineered Escherichia coli DH5a, and a positive cloning plasmid was screened and extracted. Copy number was converted according to the formula, and a positive control of CSFV with known copy number was obtained.
[37] (2) At NCBI, the sequence of the p72 gene of the ASFV was aligned, and a fragment with conserved sequence and containing the foregoing primers was selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis; the synthesized fragment was ligated to a cloning vector and transformed into genetically engineered E. coli DH5a, and a positive cloning plasmid was screened and extracted; copy number was converted according to the formula, and a positive control of ASFV with known copy number was obtained.
[38] The third step was to optimize fluorescent duplex PCR systems and amplification conditions for detecting the 5'-UTR gene of the CSFV and the p72 gene of the ASFV:
[39] (1) The concentrations of the primers and labeled probes determined in step 1 were diluted to final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 pmol/L with sterile water, respectively.
[40] (2) Using One Step PrimeScript RT-PCR Kit and recommended amplification conditions, different concentration combinations of the primers and labeled probes were subjected to fluorescence RT-PCR on an ABI 7500 PCR system and screened by the matrix method.
[41] (3) Taking an acquired minimal Ct value, a higherARn (an increased fluorescence intensity value), and a typical S-shaped amplification curve as comprehensive judgment criteria, the optimal final concentrations of forward and reverse primers for detecting the 5'-UTR gene of the CSFV were all finally determined to be 0.4 pmol/L, and the optimal final concentration of the probe was 0.6 pimol/L; the optimal final concentrations of forward and reverse primers for detecting the p72 gene of the ASFV were all 0.6 pmol/L, and the optimal final concentration of the probe was 0.6 mol/L.
[42] (4) Under conditions of the optimal concentrations of the primers and probes and the universal fluorescence RT-PCR amplification reagents, the addition amounts of reaction buffer in the amplification system were 6, 8, 10, and 12 pL successively; the amount of the reaction buffer was screened, and the amount of the reaction buffer was finally determined to be 10 L by taking an acquired minimal Ct value, a higher ARn (an increased fluorescence intensity value), and a typical S shaped amplification curve as comprehensive judgment criteria.
[43] (5) Under conditions of the optimal concentrations of the primers and probes and the optimal addition amount of the reaction buffer and universal fluorescence RT-PCR amplification reagents, the addition amounts of enzymes in the amplification system were 0.6, 0.8, 1.0, and 1.2 iL successively; the amount of the enzymes was screened, and the addition amount of the enzymes was finally determined to be 1.0 pL by taking an acquired minimal Ct value, a higher fluorescence intensity added value (ARn), and a typical S-shaped amplification curve as comprehensive judgment criteria.
[44] (6) Under conditions of the optimal concentrations of the primers and probes,the optimal amounts of reaction buffer, enzymes and universal fluorescence RT-PCR amplification reagents, the temperatures of annealing and extension were screened between 55°C and 62°C, and the temperatures of annealing and extension were finally determined to be at 60°C.
[45] (7) In an optimized 20 IL PCR system, the final concentrations of both forward and reverse primers of the CSFV were 0.4 pmol/L, and the final concentration of the probe was 0.6 pmo/L; the final concentrations of both forward and reverse primers of the ASFV were 0.6 pmol/L, and the final concentration of the probe was 0.6 pmol/L; the amount of the reaction buffer was 10 pL, the addition amount of the enzymes was 1.0 pL, and the addition amount of templates was 2.0 pL, the system was made up to a total volume of 20pL with deionized water. Optimized PCR amplification conditions were: 42°C for 5 min, 95°C for 10 s, 95°C for 5 s and 60°C for 30 s, 45 cycles.
[46] (8) Interpretation of results
[47] 1) Quality control standards:
[48] The positive control appears a specific S-shaped amplification curve, and the Ct value is around 20-25; the negative control appears no amplification curve, and there is no Ct value.
[49] If the above conditions are met, the detection results are determined to be valid.
[50] 2) Interpretation of results:
[51] A. If a sample to be tested appears specific amplification curves in both FAM and VIC channels and the Ct value is <38, it can be determined that the nucleic acids of both CSFV and ASFV are present in the sample;
[52] B. If a sample to be tested appears specific amplification curves in one of FAM or VIC channels and the Ct value is <38, it can be determined that one of the nucleic acids of CSFV or ASFV is present in the sample;
[53] C. If a sample to be tested appears specific amplification curves in FAM and/or VIC channels and the Ct value is >38 and <40, repeated tests may be needed; if the repeated result is positive, positivity can be determined; otherwise, negativity can be determined;
[54] D. If a sample to be tested appears no specific amplification curve in both FAM and VIC channels and the Ct value is >40 or there is no Ct value, it can be determined that the nucleic acid of neither CSFV nor ASFV is present in the sample.
[55] The fourth step was to conduct specificity experiments of fluorescent duplex PCR for CSFV and ASFV:
[56] (1) PRRSV, PRV, FMDV, PCV2, PPV, JEV, PEDV, and porcine TGEV were all commercial vaccines purchased. The extraction of nucleic acids of the foregoing vaccines was conducted in accordance with instructions of the corresponding commercial kits.
[57] (2) Fluorescent PCR amplification was conducted by using the optimal reaction system and amplification conditions determined in step 3. Results showed that positive controls of both CSFV and ASFV appeared specific amplification curves; PRRSV, PRV, FMDV, PCV2, PPV, JEV, PEDV, porcine TGEV, and negative control appeared no specific amplification curves. Experimental results are shown in Fig. 1.
[58] The fifth step was to establish the sensitivity curve and standard curve of the fluorescent duplex PCR for CSFV and ASFV:
[59] (1) The positive control of the 5'-UTR gene of the CSFV was subjected to 10-fold serial dilution, followed by fluorescent PCR amplification using the optimal reaction system and amplification conditions determined in step 3. The kinetic curves of the fluorescent PCR amplification are shown in Fig. 2.
[60] (2) A standard curve was plotted according to the results of amplification curves. The resulting standard curve regression equation was: Y = -3.17X + 39.73, with a correlation coefficient (R 2) of 0.999 and an amplification efficiency of 106.8%, as shown in Fig. 3; it was indicated that the primers and probes designed had high amplification efficiency and binding rate, the optimized reaction conditions were appropriate, the positive control molecule of the 5'-UTR gene of the CSFV with 10 copies was detectable, which could be used for qualitative and quantitative detection of CSFV nucleic acid.
[61] (3) The positive control of the p72 gene of the ASFV was subjected to 10-fold serial dilution, followed by fluorescent PCR amplification using the optimal reaction system and amplification conditions determined in step 3. The kinetic curves of the fluorescent PCR amplification are shown in Fig. 4.
[62] (4) A standard curve was plotted according to the results of amplification curves. The resulting standard curve regression equation was: Y = -3.256 + 38.05, with a correlation coefficient (R 2) of 1 and an amplification efficiency of 102.8%, as shown in Fig. 5; it was indicated that the primers and probes designed had high amplification efficiency and binding rate,the optimized reaction conditions were appropriate, the positive control molecule of the p72 gene of the ASFV with copies was detectable, which could be used for qualitative and quantitative detection of ASFV nucleic acid.
[63] Example 2
[64] Use of the fluorescent duplex PCR method for detecting and identifying CSFV and ASFV.
[65] A comparison was made between the fluorescent duplex PCR method for detecting and identifying CSFV and ASFV established in the present disclosure and previously established fluorescent monoplex PCR method for CSFV and ASFV, different concentrations of recombinant plasmid combinations of the two viruses was used as clinical samples (samples were numbered as 1 to 12) and sterile water was used as a negative control, laboratory testing was conducted by using the method provided by the present disclosure and the fluorescent monoplex PCR method. Results are shown in Table 1, and Fig. 6 and Fig. 7. Results show that the fluorescent monoplex PCR for CSFV had higher fluorescence intensity of the CSFV than that of the fluorescent duplex PCR for CSFV and ASFV, and results are shown in Fig. 6 (in order to align more clearly, Fig. 6 only shows amplification results of the CSFV with plasmid combinations 7 to 12), the mean Ct value of 12 plasmid combination samples differs by 0.05 in the two methods, which is almost no difference. There is almost no difference in fluorescence intensity of ASFV between the fluorescent duplex PCR for ASFV and the fluorescent monoplex PCR for CSFV and ASFV, and results are shown in Fig. 7 (in order to align more clearly, Fig. 7 only shows amplification results of the ASFV with plasmid combinations 1 to 6); the mean Ct value of 12 plasmid combination samples differs by 0.32 in the two methods, which further falls within the normal difference range. These results indicate that there are no differences in sensitivity and specificity between the fluorescent duplex PCR method for detecting and identifying
CSFV and ASFV provided by the present disclosure and the corresponding fluorescent monoplex PCR methods, but the reaction time is shortened substantially, further winning valuable time for detection.
[66] Table 1 A comparison of Ct value results between the detection method provided by the present disclosure and the fluorescent monoplex PCR methods.
Monoplex PCR Duplex PCR Sample No. CSFV ASFV CSFV ASFV
Negative control 0 0 0 0
Plasmid combination 1 22.52 34.11 22.53 34.27
Plasmid combination 2 22.57 31.77 22.55 32.53
Plasmid combination 3 22.52 27.49 22.33 27.14
Plasmid combination 4 22.45 24.24 22.22 24.91
Plasmid combination 5 22.50 21.20 22.42 21.85
Plasmid combination 6 22.56 17.8 22.44 18.34
Plasmid combination 7 22.42 14.74 22.50 15.44
Plasmid combination 8 36.07 21.35 36.75 22.08
Plasmid combination 9 33.41 21.39 33.87 22.16
Plasmid combination 10 29.96 21.01 30.19 22.02
Plasmid combination 11 19.86 21.31 19.85 21.61
Plasmid combination 12 16.52 21.41 16.39 20.70
Mean 24.45 23.15 24.50 23.47
Claims (5)
1. A fluorescent duplex PCR reagent for classical swine fever virus (CSFV) and African swine fever virus (ASFV), wherein primers and probes that are both capable of covering all virulent strains and suitable for dual detection are designed and screened based on a 5'-untranslated region (5'-UTR) gene of CSFV and a p72 gene of ASFV, respectively, comprising two specific primer pairs and two specific probes, amplified target fragments are 94 bp and 249 bp in length, respectively, and sequences of the primers and the probes are as follows:
CSFV:
forward primer CSFV-F: 5'-TGCCCAYAGTAGGACTAG-3'
reverse primer CSFV-R: 5'-CTACTGACGACTGTCCTG-3'
probe CSFV-P: 5'-TGGCGAGCTCCCTGGGTGGTCTA-3'
ASFV:
forward primer ASFV-F: 5'-GATACCACAAGATCTGCCGT-3'
reverse primer ASFV-R: 5'-CTGCTCATGGTATCAATCTTATCG-3'
probe ASFV-P: 5'-CCACGGGAGGAATACCAACCCAGTG-3'
different fluorescence reporting groups are labeled at 5'-ends of the probes CSFV-P and ASFV P, respectively, and different fluorescence quenching groups are labeled at 3'-ends of the probes CSFV-P and ASFV-P, respectively.
2. The fluorescent duplex PCR reagent for CSFV and ASFV according to claim 1, wherein the fluorescence reporting group is selected from one of FAM and VIC, and the fluorescence quenching group is selected from one of TAMRA and BHQ; the FAM and the TAMRA are labeled at the 5' end and 3'-end of the probe CSFV-P, respectively; the VIC and the BHQ are labeled at the 5'-end and 3'-end of the probe ASFV-P, respectively.
3. A fluorescent duplex PCR kit for CSFV and ASFV, comprising the two specific primer pairs and the two specific probes according to claim 1 or 2, and further comprising a reaction buffer, enzymes, a positive control, and a negative control.
4. The fluorescent duplex PCR kit for CSFV and ASFV according to claim 3, wherein the reaction buffer is One Step RT-PCR Buffer; the enzymes are Ex Taq and PrimeScript RT Enzyme; the positive control is an in vitro recombinant plasmid of a synthetic fragment of CSFV and ASFV; the negative control is sterile deionized water.
5. A fluorescent duplex PCR method for CSFV and ASFV, comprising the following steps: step 1, extracting DNA and RNA from a sample to be tested, and reverse transcribing the RNA into cDNA; step 2, with the DNA and the cDNA in step 1 as templates, using the kit according to claim 4 for fluorescent duplex PCR amplification to obtain an amplified PCR product; and step 3, analyzing the amplified PCR product, and detecting whether the CSFV and/or the ASFV are present in the sample; wherein in step 2, the optimum final concentrations of both forward primer CSFV-F and reverse primer CSFV-R for detecting the 5'-UTR gene of the CSFV are 0.4 Pmol/L, and the optimum final concentration of the probe CSFV-P is 0.6 pmol/L; the optimum final concentrations of both forward primer ASFV-F and reverse primer ASFV-R for detecting the p72 gene of the ASFV are 0.6 pmol/L, and the optimum final concentration of the probe ASFV-P is 0.6 pmol/L; in step 2, the fluorescent duplex PCR amplification has the following reaction program: 42°C for 5 min, 95°C for 10 s; 95°C for 5 s and 60°C for 30 s, 45 cycles.
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| CN114214459A (en) * | 2021-12-22 | 2022-03-22 | 山东省动物疫病预防与控制中心 | African swine fever virus and porcine circovirus type 2 dual digital PCR detection primer composition and detection method thereof |
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| CN114214459A (en) * | 2021-12-22 | 2022-03-22 | 山东省动物疫病预防与控制中心 | African swine fever virus and porcine circovirus type 2 dual digital PCR detection primer composition and detection method thereof |
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