CN111455101A - Real-time fluorescent quantitative PCR kit for detecting porcine circovirus - Google Patents

Real-time fluorescent quantitative PCR kit for detecting porcine circovirus Download PDF

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Publication number
CN111455101A
CN111455101A CN202010271723.0A CN202010271723A CN111455101A CN 111455101 A CN111455101 A CN 111455101A CN 202010271723 A CN202010271723 A CN 202010271723A CN 111455101 A CN111455101 A CN 111455101A
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quantitative pcr
porcine circovirus
fluorescent quantitative
real
primer
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李俊
田瑶
时建立
彭喆
徐绍建
李琛
吴晓燕
王硕
刘畅
韩红
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Institute Animal Science and Veterinary Medicine of Shandong AAS
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Institute Animal Science and Veterinary Medicine of Shandong AAS
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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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]

Abstract

The invention provides a real-time fluorescent quantitative PCR kit for detecting porcine circovirus, which comprises 1 upstream primer, 3 downstream primers and SYBR Green I dye, and can simultaneously detect PCV1, PCV2 and PCV 3. The primer and the kit provided by the invention have good detection specificity and high sensitivity, and are suitable for accurately and rapidly detecting the porcine circovirus types 1, 2 and 3 in a laboratory or clinic.

Description

Real-time fluorescent quantitative PCR kit for detecting porcine circovirus
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a real-time fluorescent quantitative PCR reagent for detecting porcine circovirus, in particular to a real-time fluorescent quantitative PCR of porcine circovirus type 1, 2 and 3 SYBR Green I.
Background
Porcine Circovirus (PCV) is a non-enveloped circular single-stranded DNA virus belonging to the genus circovirus of the family circovirus. Is also the smallest animal virus found to date to be capable of autonomous replication, with a genome of about 2 Kb. Before 2016, only 2 serotypes of porcine circovirus type 1 (PCV 1) and porcine circovirus type 2 (PCV 1) are found in the world, and only PCV2 is considered to be pathogenic, is a main pathogen causing Postweaning Multisystemic Wasting Syndrome (PMWS), is closely related to diseases such as Porcine Dermatitis and Npatholysis Syndrome (PDNS) and necropolizing pneumonia, and causes huge loss to the pig industry in the world. Porcine circovirus type 3 (PCV 3) was detected by scientists at the university of Kansas, Li in the USA in 2015 by genome sequencing from pigs with dermatitis nephrotic syndrome and reproductive disorders, and the genome length was 2000 bp. PCV3 infection has been reported to be associated with cardiac and multi-system inflammation, PDNS and reproductive failure. Subsequently, Fan et al, Zheng et al, Ku et al, Hehuili et al are successively found in Hubei, Shandong, Guangxi, etc. of China, but the sources and pathogenicity of Fan et al are not clear at present.
The PCR method has the advantages of simplicity, convenience and rapidness, and can be well used in PCV detection, but the defects of false positive, PCR pollution and the like exist in conventional PCR, and virus amount is obviously different in swine herds with clinical morbidity and subclinical infection due to the extensive existence of PCV, so that the conventional PCR is easy to cause missed detection. The PCV detected by the in-situ hybridization technology has good specificity, but the method cannot quantify and monitor the reaction process in real time. SYBR Green I is a fluorescent dye combined with double-stranded DNA, can be combined with a double-stranded PCR product to release a fluorescent signal during quantitative PCR reaction, is monitored and detected by an instrument system in real time, has the advantages of simple and convenient operation, high sensitivity, good repeatability, less pollution, short time consumption, automatic quantitative analysis and the like, and becomes an important method for virus detection.
Disclosure of Invention
Aiming at the problems of false positive, PCR pollution, omission and the like of the conventional PCR in the process of detecting the porcine circovirus, the invention provides a novel SYBR Green I real-time fluorescent quantitative PCR of porcine circovirus types 1, 2 and 3, which has good specificity, high sensitivity and low cost.
In order to achieve the purpose, the invention adopts the following technical scheme.
A real-time fluorescent quantitative PCR primer for detecting porcine circovirus type 1, 2 or 3 comprises an upstream primer shown as SEQ ID NO. 1 and a primer
A downstream primer of the type 1 porcine circovirus as shown in SEQ ID NO. 2; or
A downstream primer of porcine circovirus type 2 as shown in SEQ ID NO. 3; or
The downstream primer of the porcine circovirus type 3 shown as SEQ ID NO. 4.
A real-time fluorescent quantitative PCR kit for detecting porcine circovirus type 1, type 2 or type 3 comprises the primer.
The real-time fluorescent quantitative PCR kit also comprises SYBR Green I dye.
The invention has the following advantages:
according to the invention, 1 pair of specific primers are designed and respectively synthesized in a highly homologous conserved region according to the whole genome sequence of PCV1, PCV2 and PCV3, and the SYBR Green I fluorescent quantitative PCR method of PCV1, PCV2 and PCV3 is established. The invention only adopts one upstream primer and 3 downstream primers, and adopts a common upstream primer for PCV1, PCV2 and PCV3, so that the test operation is simpler, quicker and simpler. Compared with the fluorescent quantitative PCR by a probe method, the SYBR Green I fluorescent quantitative PCR does not need to design a probe, only needs a primer, and saves the cost; the method needs short time, only needs about 2 hours from sample collection to result judgment, and the method implements complete closed-tube operation, thereby fundamentally avoiding the problems of amplification product pollution and false positive existing in the conventional PCR method. The primer and the kit provided by the invention have the advantages of high detection sensitivity, high specificity and good repeatability, and can be used for epidemiological investigation, rapid detection of clinical samples, early detection, quantitative detection and later research of PCV1, PCV2 and PCV3 infection.
Drawings
FIG. 1 shows PCR amplification results of PCV1, PCV2 and PCV3 standard plasmids, wherein M is Marker 2000, 1 is PCV1 product, 2 is PCV2 product, 3 is PCV3 product, and 4 is negative control;
FIG. 2 is a fluorescent quantitative PCR standard curve of PCV1, PCV2 and PCV3, wherein a is PCV1, b is PCV2, and c is PCV 3;
FIG. 3 is fluorescence quantitative PCR melting curves of PCV1, PCV2 and PCV3, wherein 1 is PCV3, 2 is PCV1, and 3 is PCV 2;
FIG. 4 shows the PCR specificity test of PCV1, PCV2 and PCV3, wherein 1 is PCV1, 2 is PCV2, and 3 is PCV 3;
FIG. 5 is a fluorescent quantitative PCR sensitive assay for PCV,
a is PCV1, and the concentration of 1-6 is 1.00 × 107、1.05×106、8.58×104、1.11×103、1.00×102、4.04×101Copy/. mu. L, 7 is a negative control,
b is PCV2, and the concentration of 1-7 is 1.50 × 107、1.78×106、1.02×105、9.75×103、7.75×102、3.02×102、1.97×101Copy/. mu. L, 8 is a negative control,
c is PCV3, and the concentration of 1-7 is 9.30 × 10 respectively6、1.18×106、1.00×105、1.01×104、9.98×102、1.87×102、2.24×101Copy/. mu. L, 8 is a negative control;
FIG. 6 is a diagram of a conventional PCR sensitivity assay in which,
a is PCV1, and the concentration of 1-6 is 1.26 × 108、1.00×107、1.05×106、7.02×104、8.58×103、1.11×103Copy/. mu. L, 7 as negative control, M as Marker 2000,
b is PCV2, and the concentration of 1-6 is 9.84 × 107、1.50×107、1.78×106、1.02×105、9.75×103、7.75×102Copy/. mu. L, 7 as negative control, M as Marker 2000,
c is PCV3, and the concentration of 1-5 is 9.30 × 10 respectively6、1.18×106、1.00×105、1.01×104、9.98×102Copy/. mu. L, 6 is a negative control and M is Marker 2000.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 construction of the vector
1.1 primer design and Synthesis
According to the ORF1 gene sequences of PCV1, PCV2 and PCV3 in Genbank, 1 pair of specific primers (Table 1) were designed by Primer 5.0 software, and the primers were synthesized by Shanghai Biotechnology GmbH.
TABLE 1 PCV1, PCV2, PCV3 primers
Figure DEST_PATH_IMAGE001
。
1.2 preparation of Standard template
The plasmid T-PCV1, pEASY-Blunt-PCV 2 and pEASY-Blunt-PCV3 of the porcine circovirus types 1, 2 and 3 are constructed by referring to the methods in the articles of the construction of a Taq Man fluorescent quantitative PCR detection method of the porcine circovirus type I, the construction of a SYBR Green I real-time fluorescent quantitative PCR detection method of the porcine circovirus type 2, the construction of a SYBR Green I real-time fluorescent quantitative PCR detection method of the porcine circovirus type 3, and the like, and are stored at the temperature of minus 80 ℃ for later use.
Extracting recombinant plasmids from 3 subtype porcine circovirus complete genome clone plasmids T-PCV1, pEASY-Blunt-PCV 2 and pEASY-Blunt-PCV3 by using a plasmid miniprep kit, carrying out PCR amplification on the recombinant plasmids by using primer pairs in a table 1, and carrying out pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30 s, annealing at 57 ℃ for 30 s, and extension at 72 ℃ for 30 s for 30 cycles; final extension at 72 deg.C for 10 min; storing at 4 ℃. The amplification product was detected by 1.0% agarose gel electrophoresis, and the results are shown in FIG. 1: specific fragments of about 164bp, 196bp and 193bp are respectively amplified. The specific fragment is sequenced, and the DNA sequencing result shows that the sequence homology of the specific fragment and the sequences of PCV1, PCV2 and PCV3 published by GenBank is 100 percent, which shows that the standard plasmids PCV1, PCV2 and PCV3 are successfully constructed.
After the positive plasmid is identified, sequencing is carried out, and the positive recombinant plasmid with correct sequencing is respectively measured for OD in an ultramicro spectrophotometer260And OD280It was determined that the concentrations of PCV1, PCV2 and PCV3 were 224.5 mg/L, 110.4 mg/L and 295 mg/L, respectively, and the copy numbers were 4.30 × 10, respectively10Copies/. mu. L, 1.77 × 1010Copies/. mu. L, 5.38 × 1010Copy/. mu. L, the recombinant plasmid copy number was diluted to 1010An order of magnitude. The positive recombinant plasmids were diluted 10-fold, 101、102、103、104、105、106、107、108、109A total of 9 dilutions of recombinant plasmid were used as standard templates.
1.3 establishment of fluorescent quantitative PCR method
The total volume of the reaction was 25. mu. L, 2 × SYBR therein®Green Pro Taq HS Premix12.5 mu L, upstream and downstream primers 1.0 mu L, plasmid template 1.5 mu L, and ultra-pure water 25 mu L, while negative control without plasmid template is provided, the reaction program is 95 DEG CPre-denaturation for 30 s; denaturation at 95 ℃ for 5 s; annealing at 62 ℃ for 30 s, extending at 72 ℃ for 10 s, amplifying for 40 cycles, and selecting 102-106Copy/. mu. L plasmid was amplified using Roche L ightCycle®480II analysis was performed with the software to establish an amplification standard curve and a dissolution curve, as shown in FIGS. 2 and 3, respectively:
PCV1 has a slope of-3.096, an intercept 38.846, and a correlation coefficient of 0.9992, so that a linear relationship curve expression between copy (x) and cycle threshold (Ct) can be derived: ct = -3.096 lgx + 38.846. PCV2 has a slope of-3.356, an intercept of 39.513, and a correlation coefficient of 0.9999, so that a linear relationship between copy (x) and cycle threshold (Ct) can be expressed: ct = -3.356 lgx + 39.513. PCV3 has a slope of-3.611, an intercept of 40.783, and a correlation coefficient of 0.9998, so that a linear relationship between copy (x) and cycle threshold (Ct) can be expressed: ct = -3.611 lgx + 40.783;
PCV1 has a melting temperature of about 84 ℃; PCV2 has a melting temperature of about 85 ℃; PCV3 has a melting temperature of approximately 82 ℃. The negative control has no melting point peak, the standard substance amplification is single peak, and the coincidence degree of the peak is high, which indicates that the test has no pollution and the obtained data has high reliability.
Example 2 method verification
2.1 specificity test
Nucleic acids of infectious materials of Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine pseudorabies virus (PRV), Porcine Parvovirus (PPV), PCV1, PCV2 and PCV3 are respectively extracted, the RNA viruses are reversely transcribed into cDNA, real-time fluorescence quantitative PCR reaction is carried out according to the conditions in the example 1, and negative and positive controls are arranged at the same time, and the results are shown in figure 4: only PCV1, PCV2 and PCV3 have fluorescence signals, and other samples such as PRV, PPV, PRRSV, CSFV and negative control do not generate fluorescence signals, which indicates that the method has good specificity.
2.2 sensitivity test
The recombinant plasmid standard of example 1 was diluted to 10 by 10-fold gradient1、102、103、104、105、106、107、108、109The results of comparing the sensitivity differences of 2 methods by performing conventional PCR with the same primers at the same time are shown in FIG. 5 and FIG. 6, wherein the lower detection limits of the fluorescent quantitative PCR method are PCV 1: 40.3 copies/. mu. L, PCV 2: 19.7 copies/. mu. L, and PCV 3: 22.4 copies/. mu. L, respectively, and the lower detection limits of the conventional PCR method are PCV 1: 1.11 × 10, respectively3Copy/. mu. L, PCV 2: 1 × 103Copy/. mu. L, PCV 3: 9.98 × 102Copy/mu L, which shows that the real-time fluorescent quantitative PCR method of the invention has better sensitivity than the conventional PCR and higher reliability of the detection result.
2.3 repeatability test
Choose 103、104、105The diluted plasmid standard is subjected to a repeated real-time fluorescent quantitative PCR amplification test, each template of the intra-group test is repeated 3 times at the same time, the test between the groups is repeated 3 times continuously, the obtained average value, standard deviation and variation coefficient of Ct are subjected to statistical analysis, and the results are shown in tables 2-7: the result shows that the variation coefficients of the standard substance amplification of 3 dilutions are all less than 1.00%, and the method adopting the fluorescent quantitative PCR detection has high repeatability.
TABLE 2 Intra-group statistics for fluorescent quantitative PCR detection of PCV1
Figure DEST_PATH_IMAGE003
TABLE 3 intergroup statistics for fluorescent quantitative PCR detection of PCV1
Figure DEST_PATH_IMAGE005
TABLE 4 Intra-group statistics for fluorescent quantitative PCR detection of PCV2
Figure DEST_PATH_IMAGE007
TABLE 5 inter-group statistics for fluorescent quantitative PCR detection of PCV2
Figure DEST_PATH_IMAGE009
TABLE 6 Intra-group statistics for fluorescent quantitative PCR detection of PCV3
Figure DEST_PATH_IMAGE011
TABLE 7 inter-group statistics for fluorescent quantitative PCR detection of PCV3
Figure DEST_PATH_IMAGE013
。
2.4 clinical sample testing
100 suspected PCV pathogens are taken and respectively subjected to SYBR Green I fluorescent quantitative PCR and conventional PCR detection to compare the sensitivity of the 2 methods. As a result, the sensitivity of the fluorescent quantitative PCR was higher than that of the conventional PCR, as shown in Table 8.
TABLE 8 statistics of fluorescence quantitative PCR clinical test results
Figure DEST_PATH_IMAGE015
。
Sequence listing
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<120> real-time fluorescent quantitative PCR kit for detecting porcine circovirus
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Claims (3)

1. A primer for detecting real-time fluorescent quantitative PCR of porcine circovirus type 1, 2 or 3 is characterized in that the primer comprises an upstream primer shown as SEQ ID NO. 1 and a primer
A downstream primer of the type 1 porcine circovirus as shown in SEQ ID NO. 2; or
A downstream primer of porcine circovirus type 2 as shown in SEQ ID NO. 3; or
The downstream primer of the porcine circovirus type 3 shown as SEQ ID NO. 4.
2. A real-time fluorescent quantitative PCR kit for detecting porcine circovirus type 1, type 2 or type 3, comprising the primer of claim 1.
3. The kit of claim 2, wherein the real-time fluorescent quantitative PCR kit further comprises SYBR Green I dye.
CN202010271723.0A 2020-01-16 2020-04-09 Real-time fluorescent quantitative PCR kit for detecting porcine circovirus Pending CN111455101A (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN107916303A (en) * 2017-12-28 2018-04-17 广州维佰生物科技有限公司 Quick HRM detection primers, kit and the method for differentiating 1 type of pig circular ring virus, 2 types and 3 types
CN109593883A (en) * 2017-09-30 2019-04-09 洛阳普莱柯万泰生物技术有限公司 Kit of the pig circular ring virus multiple real time fluorescence PCR detection primer to, probe and preparation
CN109762943A (en) * 2019-03-22 2019-05-17 福建省农业科学院畜牧兽医研究所 For identifying the primer sets and its application of 1,2,3 type of pig circular ring virus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109593883A (en) * 2017-09-30 2019-04-09 洛阳普莱柯万泰生物技术有限公司 Kit of the pig circular ring virus multiple real time fluorescence PCR detection primer to, probe and preparation
CN107916303A (en) * 2017-12-28 2018-04-17 广州维佰生物科技有限公司 Quick HRM detection primers, kit and the method for differentiating 1 type of pig circular ring virus, 2 types and 3 types
CN109762943A (en) * 2019-03-22 2019-05-17 福建省农业科学院畜牧兽医研究所 For identifying the primer sets and its application of 1,2,3 type of pig circular ring virus

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* Cited by examiner, † Cited by third party
Title
KELI YANG等: "Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens", 《BMC INFECTIOUS DISEASES》 *
羊泽晓等: "猪圆环病毒多重PCR检测方法的建立", 《西北农业学报》 *

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Application publication date: 20200728