CN111763771A - Quadruple real-time fluorescent PCR (polymerase chain reaction) identification and detection method for porcine circovirus types 1 to 4 - Google Patents
Quadruple real-time fluorescent PCR (polymerase chain reaction) identification and detection method for porcine circovirus types 1 to 4 Download PDFInfo
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Abstract
A quadruple real-time fluorescent PCR identification and detection method for porcine circovirus types 1 to 4 belongs to the technical field of biology. The invention designs specific primers and probe sequences capable of identifying PCV1, PCV2, PCV3 and PCV4 aiming at ORF1 and ORF2 genes of PCV of different genotypes, probes of PCV1-4 strains marked by different fluorescein are used, each probe can be marked by any one fluorescein, the four probes are marked by the fluorescein detected by different detection channels, the primers and the probes which are respectively used for detecting the PCV1-4 strains or the primers and the probes for identifying the PCV1-4 strains are placed in the same reaction tube, a fluorescence PCR amplification instrument is used for real-time fluorescence PCR reaction, and the identification and detection of the PCV1-4 strains can be completed by carrying out detection operation on the same sample once.
Description
Technical Field
The invention belongs to the technical field of biology, relates to quadruple real-time fluorescent PCR detection, and particularly relates to a quadruple real-time fluorescent PCR identification and detection method for porcine circovirus types 1 to 4.
Background
Porcine Circovirus (PCV) belongs to the family circoviridae, the genus circovirus. PCV is the smallest currently known circular single-stranded DNA virus with a virion diameter of about 17nm, without an envelope, and with icosahedral symmetry. Currently, PCV can be classified into four genotypes (PCV1, PCV2, PCV3, PCV 4). In 1974, PCV1 was first discovered from a contaminating porcine kidney cell line PK-15. PCV1 is currently considered to be nonpathogenic to pigs. In 1998, PCV2 was first reported to be closely related to postweaning multi-system failure syndrome, porcine dermatitis nephrosis syndrome and the like. In 2015, PCV3 was first discovered in porcine dermatitis nephrosis syndrome and aborted swine herds. In 2019, PCV4 is found in Hunan diseased swinery in China for the first time. The nucleotide homology among the four genotypes of PCV is lower and is 35 to 75 percent. As the pathogenicity, antigenicity and genome of PCV strains have obvious difference, the quick differential diagnosis of PCV strains of different subtypes has important significance for taking targeted effective prevention and control measures clinically.
The current classical methods for detecting PCV mainly comprise serological detection, PCR detection and the like. However, these methods are time consuming and labor intensive, have low sensitivity and are prone to false negatives. At present, although real-time fluorescent PCR detection methods aiming at different PCV subtypes are established, the four genotypes of viruses from PCV1 to PCV4 cannot be simultaneously identified and detected. Therefore, the invention aims to establish a quadruple differential detection method from PCV1 to PCV4, which is rapid, simple, strong in specificity, high in sensitivity and good in repeatability.
Disclosure of Invention
The invention provides a quadruple real-time fluorescent PCR (polymerase chain reaction) identification and detection method for porcine circovirus type 1 to type 4 (PCV1-4) in order to make up the defects of the quick identification and detection method for four genotype strains of PCV. The detection method can judge whether the sample contains PCV1, PCV2, PCV3, PCV4 or the common infection of more than two genotype strains by only one detection. The method is rapid, convenient, high in specificity, high in sensitivity and good in repeatability, can be used for rapid identification and detection analysis of PCV infection conditions of large-batch samples, and has a good application prospect.
The invention is realized by the following technical scheme:
all nucleotide sequences listed herein are written from 5 'to 3' end.
One aspect of the invention relates to four primer pairs (comprising eight primer sequences), wherein:
the PCV1 primer pair comprises PCV1-F2020: AACCCCATAAGAGGTGGGTGTT and PCV 1-R2020: TTCTACCCTCTTCCAAACCTTCCT;
the PCV2 primer pair comprises PCV2-F2020: CTGAGTCTTTTTTATCACTTCGTAATGGT and PCV 2-R2020: ACTGCGTTCGAAAACAGTATATACGA;
the PCV3 primer pair comprises PCV3-F2020: CATAAATGCTCCAAAGCAGTGCT and PCV 3-R2020: TCACCCAGGACAAAGCCTCTT;
the PCV4 primer pair comprises PCV4-F2020:
ATTATTAAACAGACTTTATTTGTGTCATCACTT and PCV 4-R2020: ACAGGGATAATGCGTAGTGATCACT in a pharmaceutically acceptable carrier.
Another aspect of the invention relates to four probes comprising:
probe PCV1-P2020 of PCV 1: TCCGAGGAGGAGAAAAACAAAATACGGGA;
probe PCV2-P2020 of PCV 2:
TTAAGTGGGGGGTCTTTAAGATTAAATTCTCTGAATTGT;
probe PCV3-P2020 of PCV 3: ATATGTGTTGAGCCATGGGGTGGGTCT;
probe PCV4-P2020 of PCV 4:
ATACTACACTTGATCTTAGCCAAAAGGCTCGTTGA in a pharmaceutically acceptable carrier.
The invention also relates to a composition of an oligonucleotide primer pair and a probe for simultaneously detecting PCV 1-type 4 (PCV1-4) strains, which comprises the primer pair and the probe.
In addition, the composition can be prepared into a kit.
Furthermore, the invention also relates to the application of the kit in simultaneously detecting PCV 1-type 4 (PCV1-4) strains, wherein the probe is labeled by any one fluorescein, and the four probes are labeled by the fluorescein using different detection channels, and all the four probes can be selected from the fluorescein of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 and the like.
A quadruple real-time fluorescence PCR method for simultaneously identifying and detecting PCV1 to type 4 (PCV1-4) by using the composition comprises the following steps:
(1) specific primer and probe sequences capable of identifying PCV1, PCV2, PCV3 and PCV4 are designed aiming at ORF1 and ORF2 genes of PCV with different genotypes.
(2) The probes of PCV1-4 strain labeled by different fluorescein can be labeled by any one fluorescein per probe, and the four probes are labeled by the fluorescein which can be detected by different detection channels and can be selected from the fluorescein of FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 and the like.
(3) The primer and the probe which are respectively used for detecting the PCV1-4 strain or the primer and the probe which are used for identifying the PCV1-4 strain are placed in the same reaction tube, and a real-time fluorescent PCR reaction is carried out by using a fluorescent PCR amplification instrument.
The invention has the remarkable characteristics that: the high-efficiency amplification performance of the PCR technology, the good specificity of the nucleic acid hybridization technology and the quick sensitivity of the fluorescence detection technology are fully utilized, the identification and detection of PCV1 to type 4 (PCV1-4) strains can be completed by carrying out detection operation on the same sample once, and the infection of which PCV genotype strain the sample is or the common infection of more than two PCV different genotype strains can be determined. The method has the advantages of rapidness, convenience, strong specificity, high sensitivity, good reliability, reduction in detection cost, improvement in detection efficiency and the like.
Drawings
FIG. 1 is a specific amplification curve diagram of a quadruple real-time fluorescent PCR detection method for PCV1 to type 4 (PCV1-4) strains; in the figure, PCV1, PCV2, PCV3 and PCV4 representative strains can obtain specific amplification curves, while other viruses (PPV, PRV, PRRSV, PEDV and CSFV) are not specifically amplified;
FIG. 2 is a sensitive amplification curve diagram of PCV1-4 strain in a quadruple real-time fluorescent PCR detection method of PCV1 to PCV type 4 (PCV1-4) strain;
in the graph, A is a curve representing PCV1 at a concentration of 1x108～1x101Amplification results in copy number; the curves in FIG. B represent PCV2 at concentrations of 1X10, respectively8～1x101Amplification results in copy number; the C curves in the graph represent PCV3 at a concentration of 1x108～1x101Amplification results in copy number; the D curves in the graph represent PCV4 at a concentration of 1x108～1x101Amplification results in copy number.
Detailed Description
For routine experimentation in the following examples, see molecular cloning, A laboratory Manual, third edition, by Sambrook et al (Beijing: scientific Press, 2002), the use of the instrument is described with reference to instructions for the operation of the instrument.
The actual sources and specifications of the organisms used in the present invention are as follows:
since PCV3 and PCV4 cannot be isolated in vitro at present, the pathogens used in the examples of the present invention were all sequencing-verified clinically positive samples. Positive samples of porcine circovirus type 1 (PCV1), type 2 (PCV2), type 3 (PCV3), type 4 (PCV4), Porcine Parvovirus (PPV), porcine pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV) and the like are all preserved in the laboratory.
In the examples of the present invention, other reagents used: RNase Free H2O was purchased from Solambio; HiPureTissue DNA Mini Kit was purchased from magenta Meiji Bio; 2 × Premix Ex Taq, 2 × PrimeSTAR MAXDNA Polymearse from TAKARA; FastPure Plasmid Mini Kit was purchased from Novovozan Biotech Co., Ltd; DNA Marker was purchased from Zhejiang Bo and jin science, Inc. The primers and positive standard plasmids used in the assay were both synthesized by Suzhou Jinweizhi Biotech, Inc., and the probes were synthesized by Kunshan Ponopol and Biotech, Inc.
Example 1:
establishment and evaluation of quadruple real-time fluorescent PCR (polymerase chain reaction) identification and detection method for porcine circovirus type 1 to type 4 (PCV1-4)
1.1 design of primers and probes
According to the PCV strain whole genome obtained from GenBank database, sequence alignment software DNAMAN is used for multi-sequence alignment analysis, and 4 specific TaqMan probes and 4 pairs of primers for identifying PCV1, PCV2, PCV3 and PCV4 are respectively designed based on highly conserved regions in ORF1 and ORF2 genes. The 5' ends of the probes are respectively marked with different fluorescent groups (FAM, HEX, ROX and TAMRA). The primers and probes in PCV quadruplex real-time fluorescent PCR are shown in Table 1-1.
TABLE 1-1 primers and probes for construction of PCV quadruple real-time fluorescent PCR identification detection method
The positions of the corresponding primers and probes in the genome were determined for PCV1 SC-1 strain (DQ358813), PCV2SD17-36 strain (MH191378), PCV 329160 strain (KT869077), and PCV4 HNU-AHG1-2019 strain (MK986820), respectively.
1.2 plasmid Standard Synthesis
The PCV quadruple real-time fluorescent PCR standard plasmid synthesis method comprises the following steps:
sequences obtained by amplifying PCV1, PCV2, PCV3 and PCV4 by 4 pairs of specific primers of quadruple PCV real-time fluorescent PCR are spliced and inserted into a pUC57 vector to prepare a standard plasmid. The standard quality grain synthesis is completed by Suzhou Jinwei Zhi, and the concentration is measured by a micro spectrophotometer and stored to-20 ℃ after the sequencing verification.
1.3DNA extraction
Viral DNA was extracted from the diseased pig Tissue samples with reference to the HiPure Tissue DNA Mini Kit by the following method:
(1) 20mg of tissue sample was placed in an EP tube, and 220. mu.L Buffer ATL and 20. mu.L protease K were added and vortexed to mix. Digestion was carried out overnight at 55 ℃ with inversion during several times.
(2) Add 10. mu.L RNase A to the digest, mix it by inversion, and leave it at room temperature for 10 min.
(3) Add 250. mu.L Buffer DL to the digest, vortex at high speed for 10sec, and water bath at 70 ℃ for 10 min.
(4) Add 250. mu.L of absolute ethanol to the digest and vortex for 15 sec.
(5) Liquid on the tube wall was collected by brief centrifugation.
(6) Loading the DNA binding column into a collection tube, and transferring the mixed solution obtained in the step 5 to the column. Centrifuge at 10,000 Xg for 1 min.
(7) The filtrate was decanted, 500. mu.L of Buffer GW1 was added, and the mixture was centrifuged at 10,000 Xg for 1 min.
(8) The filtrate was decanted, 650. mu.L of Buffer GW2 was added, and the mixture was centrifuged at 10,000 Xg for 1 min.
(9) And repeating the operation of the step 8.
(10) The filtrate was decanted, the column was reloaded into the collection tube and centrifuged at 10,000 Xg for 2 min.
(11) The column was loaded in a new 1.5mL EP tube, 10-100. mu.L of Buffer TE preheated to 70 ℃ was added to the center of the membrane of the column, and left to stand for 3 min. Centrifuge at 10,000 Xg for 1 min.
(12) And repeating the operation of the step 11.
(13) The DNA binding column was discarded, DNA absorbance and concentration were determined and stored at-20 ℃.
1.4 preparation of standards
The plasmid concentration was converted to RNA copy number according to the following formula:
y(copies/μL)＝(6.02×1023)×[(x ng/uL)×10-9DNA]/(DNA length×660)
use of RNase Free H2Dilution of plasmid to copy number 109copies/. mu.L stock solution, 10-fold serial dilutions of plasmid standards, selected at a concentration of 101–108Plasmid standards of copies/. mu.L, according to the primers and probes in Table 1-1, Real-Time fluorescent PCR reaction was performed using the StepOne Plus Real-Time PCR system to draw a standard curve for PCV quadruple Real-Time fluorescent quantitative PCR. The PCV quadruple real-time fluorescent PCR reaction system is shown in the following tables 1-2:
TABLE 1-2 PCV quadruple real-time fluorescent PCR reaction system
| Reaction solution | Volume of | |
| 2×Premix | 10μL | |
| 4 upstream and downstream primer mixed liquor (10 mu M) | 0.4μL/0.4μL/0.4μL/0.4 |
|
| 4 kinds of probes | 0.2μL/0.2μL/0.2μL/0.2μL | |
| DNA template | 2μL | |
| RNase Free H2O | Make up to 20 mu L |
The PCR reaction program is: 30s at 95 ℃; 5s at 95 ℃, 1min at 60 ℃ and 40 cycles.
1.5 specificity test
The DNA of PCV (PCV1, PCV2, PV3 and PCV4) of different genotypes and the nucleic acids of CSFV, PEDV, PRRSV, PRV and PPV were detected by the PCV quadruple real-time fluorescence quantitative PCR method established at 1.4 to evaluate the specificity of the method. As shown in FIG. 1, when PCV of 4 genotypes is detected by a PCV quadruple real-time fluorescent PCR method, specific FAM, HEX, ROX and TAMRA fluorescent signals can be detected; when the quadruple real-time fluorescence PCR method is used for detecting CSFV, PEDV, PRRSV, PRV and PPV, no specific fluorescence signal is generated, which shows that the method has good specificity.
1.6 sensitivity test
The copy number detected by the PCV quadruple real-time fluorescence quantitative PCR method established by 1.4 is 101–108As shown in FIG. 2, the detection limits of PCV1, PCV2, PCV3 and PCV4 in the PCV quadruple real-time fluorescent PCR are all 1 × 101copies/. mu.L, indicating that the method has good sensitivity. Meanwhile, the detection efficiency of each primer probe in the PCV quadruple real-time fluorescent quantitative PCR method is consistent with the detection result of the quadruple real-time fluorescent PCR, which shows that each probe has good specificity and does not generate obvious mutual interference phenomenon when being used in a combined way.
1.7 in-group and between-group reproducibility test
The copy number detected by the PCV quadruple real-time fluorescence quantitative PCR method established by 1.4 is 106、104And 102Plasmid standards of copies/. mu.L, to determine within-and between-group reproducibility. Wherein, the plasmid standard substance of each concentration is repeatedly detected for 3 times (n is 3), and the intra-group variation coefficient is calculated according to the CT value; and determining the variation coefficient among groups by detecting the plasmid standard substance according to the MIQE guidance method so as to verify the repeatability of the method. As shown in tables 1-3, the variation coefficients of the PCV quadruple real-time fluorescent PCR method in the intra-group test and the inter-group test are 0.07-2.72% and 0.54-3.91%, respectively. Indicating that the method has good reproducibility within and between groups.
TABLE 1-3 analysis of repeatability within and between groups of PCV quadruple real-time fluorescent PCR method
Intra-and inter-group repeats are all expressed as mean ± standard deviation of 3 repeats.
1.8 clinical sample testing
120 clinical sick pig samples collected from Jiangsu province in 2020 were detected 2016-. The results are shown in tables 1-4, and the positive samples of PCV1 are 6, and the positive rate is 5%; 59 positive samples of PCV2 are obtained, and the positive rate is 49.2%; 9 positive samples of PCV3, wherein the positive rate is 7.5%; the positive samples of PCV4 were 4, and the positive rate was 3.3%. All positive samples above were further confirmed by PCR amplification and Sanger sequencing. The result shows that the PCV quadruple real-time fluorescence PCR method has good clinical practicability and application prospect.
TABLE 1-42016-year-old Jiangsu sick pig sample Positive rate analysis of different PCV genotype strains
| Pathogens | 2016 | 2017 | 2018 | 2019 | 2020 | Total of |
| |
0/5* | 0/8 | 3/36 | 2/23 | 1/48 | 6/120(5.0％) |
| |
4/5 | 6/8 | 21/36 | 14/23 | 14/48 | 59/120(49.2％) |
| |
1/5 | 0/8 | 1/36 | 0/23 | 7/48 | 9/120(7.5％) |
| |
0/5 | 0/8 | 2/36 | 2/23 | 0/48 | 4/120(3.3％) |
This ratio represents the number of positive samples in all test samples.
Sequence listing
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Claims (4)
1. The quadruple real-time fluorescent PCR identification and detection method for porcine circovirus types 1 to 4 is characterized by comprising the following steps:
1) designing specific primer and probe sequences capable of identifying PCV1, PCV2, PCV3 and PCV4 aiming at ORF1 and ORF2 genes of PCV with different genotypes;
2) probes of PCV1-4 strain labeled by different fluorescein, each probe can be labeled by any one fluorescein, and the four probes are labeled by fluorescein detected by different detection channels and can be selected from FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX or CY5 fluorescein;
3) the primer and the probe which are respectively used for detecting the PCV1-4 strain or the primer and the probe which are used for identifying the PCV1-4 strain are placed in the same reaction tube, and a real-time fluorescent PCR reaction is carried out by using a fluorescent PCR amplification instrument.
2. The quadruple real-time fluorescent PCR (polymerase chain reaction) identification and detection method for porcine circovirus types 1 to 4 according to claim 1, which is characterized in that the primers are four primer pairs comprising eight primer sequences,
the PCV1 primer pair comprises PCV1-F2020: AACCCCATAAGAGGTGGGTGTT and PCV 1-R2020: TTCTACCCTCTTCCAAACCTTCCT;
the PCV2 primer pair comprises PCV2-F2020: CTGAGTCTTTTTTATCACTTCGTAATGGT and PCV 2-R2020: ACTGCGTTCGAAAACAGTATATACGA;
the PCV3 primer pair comprises PCV3-F2020: CATAAATGCTCCAAAGCAGTGCT and PCV 3-R2020: TCACCCAGGACAAAGCCTCTT;
the PCV4 primer pair comprises PCV4-F2020: ATTATTAAACAGACTTTATTTGTGTCATCACTT and PCV 4-R2020: ACAGGGATAATGCGTAGTGATCACT in a pharmaceutically acceptable carrier.
3. The method for the quadruple real-time fluorescent PCR (polymerase chain reaction) differential detection of porcine circovirus types 1 to 4 according to claim 2, wherein the probes are four probes comprising:
probe PCV1-P2020 of PCV 1: TCCGAGGAGGAGAAAAACAAAATACGGGA;
probe PCV2-P2020 of PCV 2: TTAAGTGGGGGGTCTTTAAGATTAAATTCTCTGAATTGT;
probe PCV3-P2020 of PCV 3: ATATGTGTTGAGCCATGGGGTGGGTCT;
probe PCV4-P2020 of PCV 4: ATACTACACTTGATCTTAGCCAAAAGGCTCGTTGA in a pharmaceutically acceptable carrier.
4. The quadruple real-time fluorescent PCR (polymerase chain reaction) differential detection method for porcine circovirus types 1 to 4 according to claim 3, which is characterized in that a combination of primers and probes is prepared into a kit for differential detection.
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| CN112391499A (en) * | 2020-12-04 | 2021-02-23 | 福建省农业科学院畜牧兽医研究所 | Primer group of loop-mediated isothermal amplification method for detecting porcine circovirus type 4 |
| CN112646931A (en) * | 2020-12-31 | 2021-04-13 | 四川省畜牧科学研究院 | Primer pair, probe, kit and detection method for detecting porcine circovirus type 4 |
| CN113249524A (en) * | 2021-06-25 | 2021-08-13 | 广东省农业科学院动物卫生研究所 | Triple real-time fluorescent quantitative PCR primer and probe composition for detecting various porcine circovirus and application thereof |
| CN113322352A (en) * | 2021-06-08 | 2021-08-31 | 浙江省检验检疫科学技术研究院 | Method, primer, probe and kit for detecting porcine circovirus by triple digital microdroplet PCR |
| CN113403430A (en) * | 2021-07-26 | 2021-09-17 | 浙江省动物疫病预防控制中心 | Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application |
| CN113584230A (en) * | 2021-09-01 | 2021-11-02 | 青岛易邦生物工程有限公司 | Reagent and method for detecting different genotypes of porcine circovirus |
| CN113774166A (en) * | 2021-09-13 | 2021-12-10 | 青岛农业大学 | Porcine circovirus type 2, type 3 and type 4 on-site rapid high-sensitivity differential diagnosis kit and use method thereof |
| CN114480379A (en) * | 2022-03-18 | 2022-05-13 | 达州职业技术学院 | Establishment and application of PCV-2, PCV-3 and PCV-4 triple PCR identification detection method |
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