CN116144844B - Chip type digital PCR (polymerase chain reaction) kit for detecting porcine sapelo virus - Google Patents

Chip type digital PCR (polymerase chain reaction) kit for detecting porcine sapelo virus Download PDF

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CN116144844B
CN116144844B CN202310174664.9A CN202310174664A CN116144844B CN 116144844 B CN116144844 B CN 116144844B CN 202310174664 A CN202310174664 A CN 202310174664A CN 116144844 B CN116144844 B CN 116144844B
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张越
魏战勇
祖少坡
樊晓阳
闫一博
张宇航
冯于童
徐盟龙
郭子涵
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Henan Agricultural University
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Abstract

The invention discloses a chip type digital PCR kit for detecting porcine sapelo virus, and relates to the technical field of molecular biology. The chip type digital PCR kit comprises a primer pair and a probe combination, wherein the primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3. The invention establishes a chip type digital PCR detection method for the porcine sapelo virus, and based on the method, the invention provides a chip type digital PCR kit for detecting the porcine sapelo virus, which has strong specificity for quantitative detection of the porcine sapelo virus, high sensitivity and stable repeatability, and provides important guiding significance for early diagnosis and biological safety clinical prevention and treatment of the porcine sapelo virus.

Description

Chip type digital PCR (polymerase chain reaction) kit for detecting porcine sapelo virus
Technical Field
The invention relates to the technical field of molecular biology, in particular to a chip type digital PCR kit for detecting porcine sapelo virus.
Background
Porcine sapelo virus (Porcine sapelovirus; PSV) can cause multiple system syndromes such as diarrhea, enteritis, pneumonia, reproductive dysfunction, non-suppurative encephalitis, poliomyelitis, pericarditis, myocarditis, skin injury, and nervous system disorder in pigs. In part of pig farms, the positive rate of the porcine sapelo virus is up to 60 percent, and the porcine sapelo virus is easy to be mixed with porcine delta coronavirus (PDCoV), porcine Epidemic Diarrhea Virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine Circovirus (PCV) and the like clinically. The porcine sapelo virus can reproduce in the intestinal epithelial cells of pigs, so that the villus atrophy and the reduction of bleeding and exudation of the natural layer of the small intestine can be seen, the toxin can be continuously discharged after the infected pigs are recovered, and after the pregnant sows are infected, the viremia is transmitted through blood sources, so that the embryo and the fetus are infected, the embryo death, the dead yield, the mummy embryo, the fetal malformation and the like are caused, and the virus causes serious economic loss to the pig industry. At present, the porcine sapelo virus has little research, and no commercial vaccine exists for the porcine sapelo virus, and no effective treatment measures exist, so that the porcine sapelo virus can only be mainly prevented at present. Therefore, the method is an important means for ensuring the safety of the pig industry by adopting timely early diagnosis and preventive measures and timely treatment of suspected or diseased pigs in the face of the pig sapelo virus.
The current common detection methods for the porcine sapelo virus mainly comprise methods of virus separation and identification, indirect ELISA, reverse transcription PCR, taqMan real-time quantitative PCR and the like. The virus separation and identification requires high instruments and complex operation procedures, and has certain limitation on clinical rapid diagnosis; the indirect ELISA operation method is simple, but animal serum is needed, and pathogenic molecules cannot be detected; although TaqMan real-time fluorescent quantitative PCR (RT-qPCR) is highly sensitive, this method relies heavily on Ct values and standard curves, and it is difficult to accurately detect templates in early stages of viral infection when sample concentrations are too low or inhibitors are present. Therefore, there is a need to develop a high-sensitivity detection technique for porcine sapelo virus.
Disclosure of Invention
The invention aims to provide a chip type digital PCR kit for detecting porcine sapelo virus, which solves the problems of the prior art, has strong specificity, high sensitivity and stable repeatability for quantitative detection of the porcine sapelo virus, and provides important guiding significance for early diagnosis and biosafety clinical prevention and treatment of the porcine sapelo virus.
Chip Digital PCR (cdPCR) is an analysis technology based on single-component template amplification, which can quickly and accurately divide a sample into a plurality of independent units after flowing, and realizes accurate quantification of copy number level. A chip has 20000 liquid drops, each unit contains 0, 1 and a plurality of target nucleic acid molecules, a physical partition with uniform liquid drop size can be formed, then samples are amplified in each reaction unit, after the amplification is finished, the copy number of target molecules is calculated according to the ratio of positive liquid drops to negative liquid drops according to the poisson distribution principle, and absolute quantification of target fragments with the sensitivity reaching 0.01% is realized. Breaks through the limitation of the traditional PCR detection in distinguishing smaller copy number difference, has stronger specificity and excellent repeatability, does not need to rely on the cycle threshold value of an amplification curve for quantification, does not need to use a standard curve and an internal reference gene, and is more accurate and reliable in quantifying low-concentration nucleic acid. Therefore, the present invention contemplates improving the detection sensitivity of porcine sapelo virus by establishing a cdPCR detection method of porcine sapelo virus.
According to the invention, a specific primer and a probe are designed according to a specific conserved gene sequence target PSV_BRA/UEL-WB20/135' UTR of the porcine sapelovirus, a porcine sapelovirus cdPCR detection method is established, and the established cdPCR detection method is adopted to carry out detection analysis on clinical onset samples, air samples and the like of the porcine sapelovirus, and the result shows that the minimum detection limit of the cdPCR detection method is 3.12 copies/mu L, the sensitivity is improved by about 1000 times compared with the RT-PCR detection method, and the sensitivity is improved by about 10 times compared with the TaqMan real-time fluorescence quantification (RT-qPCR) detection method.
Based on this, the present invention provides the following scheme:
the invention provides a primer pair and a probe combination for detecting porcine sapelo virus by chip type digital PCR, wherein the primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
Further, the 5 'end of the probe is connected with a FAM group, and the 3' end of the probe is connected with a BHQ1 group.
The invention also provides application of the primer pair and the probe combination in preparing a chip type digital PCR kit for detecting the porcine sapelovirus.
The invention also provides a chip type digital PCR kit for detecting the porcine sapelo virus, which comprises the primer pair and the probe combination.
Further, the concentration ratio of the primer pair and the probe is 500nM/1500nM.
Further, the chip type digital PCR kit also comprises a digital PCR reaction buffer solution, a salpeter virus positive detection template and nuclease-free water.
Further, the reaction program of the chip type digital PCR kit is as follows: pre-denaturation at 95℃for 2min, denaturation at 95℃for 15s, annealing at 54℃for 30s,45 cycles.
The invention discloses the following technical effects:
according to the invention, a specific primer and a probe are designed according to a specific conserved gene sequence target PSV_BRA/UEL-WB20/135' UTR of the porcine sapelovirus, a porcine sapelovirus cdPCR detection method is established, the annealing temperature, the concentration ratio of the primer and the probe of the method are optimized, and the sensitivity, the specificity, the clinical sample and the air sample are verified and detected. According to the swine sapelovirus cdPCR detection method, the optimal annealing temperature is 54 ℃, the optimal primer probe concentration ratio is 500nM/1500nM, the digital PCR is not required to depend on a standard curve and a Ct value, and the concentration of virus particles in a target template can be calculated in absolute quantification by only directly depending on a poisson distribution principle. The cdPCR detection method established by the invention is used for detecting and analyzing clinical onset samples, air samples and the like of the porcine sapelovirus, and the result shows that each sample has liquid drop generation, so that the operation meets the requirements, the effective liquid drop rate is about 95%, the absolute and accurate quantitative analysis technology is adopted, and the detection sensitivity can reach 0.1%. The minimum detection limit for detecting the concentration of the salmeterol virus of the pig is 3.12 copies/. Mu.L, the sensitivity is improved by about 1000 times compared with the RT-PCR detection method, and the sensitivity is improved by about 10 times compared with the RT-qPCR detection method.
The established cd PCR detection method has strong specificity, high sensitivity and stable repeatability for quantitative detection of the porcine sapelo virus, and provides important guiding significance for early diagnosis, epidemic disease monitoring and biological safety comprehensive control of the porcine sapelo virus.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of a standard plasmid of porcine sapelo virus;
FIG. 2 is a graph showing the droplet distribution of the annealing temperature optimization experiment (A) and the primer probe concentration ratio optimization experiment (B) in example 1;
FIG. 3 is a droplet distribution diagram of the specificity experiment in example 1;
FIG. 4 is a droplet distribution diagram of a sensitivity experiment in example 1;
FIG. 5 shows the experimental results of aerosol air sample detection using chip-type digital PCR in example 1.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1. Materials and methods
1. Material preparation
Comprises the following materials:
(1) Chip type digital PCR detection kit;
(2) BioDital-blue digital PCR chip;
(3) BioDital-blue digital PCR full-automatic liquid drop preparation instrument
(4) BioDital-blue digital PCR amplification instrument;
(5) A BioDital-blue digital PCR biochip reader;
(6) Upstream primer (SEQ ID NO. 1): 5'-GGTGTGCTCCATTGGTGAT-3' (Henan Shang Ya Biotechnology Co., ltd.) 10nM;
(7) Downstream primer (SEQ ID NO. 2): 5'-CCTCAATTGCACATTCCCTCTA-3' (Henan Shang Ya Biotechnology Co., ltd.) 10nM;
(8) Probe (SEQ ID No. 3): FAM-TGGTTACGGGAGAGTAGGCAGTGA-BHQ1 (Henan Shang Ya Biotechnology Co., ltd.) 10nM;
(9) The main strains: porcine Sapelo Virus (PSV), porcine delta coronavirus (porcine deltacoronavirus, PDCoV), porcine epidemic diarrhea (porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis (transmissible gastroenteritis virus, TGEV), porcine circovirus (porcine circovirus, PCV), porcine pseudorabies virus (pseudorabies virus, PRV).
48 samples of porcine diarrhea clinical tissue were collected from Henan, hebei and Shanxi farms.
RNA extraction
Extracting total RNA according to Trizol total RNA extraction instruction, and performing reverse transcription to obtain cDNA by using a Vazyme reverse transcription kit;
3. sample marking
The construction of a standard plasmid of porcine sapelo virus comprises the following specific steps: extracting total RNA of the porcine sapelovirus, reversely transcribing the total RNA into cDNA, carrying out PCR amplification according to specific conserved gene sequence primers (SEQ ID NO.1 and SEQ ID NO. 2), and connecting a PCR product to a pMD-18T vector to construct a standard plasmid which is used as a positive detection template of the porcine sapelovirus, wherein the schematic diagram of the constructed standard plasmid is shown in figure 1.
Positive samples diluted 10-fold in gradient were used to test the sensitivity of the swine sapelo virus chip type digital PCR method of the present invention.
5. Configuration of reaction System
A reaction system of 35. Mu.L was used, and 2. Mu.L of the nucleic acid template was added to each reaction, and the configuration of the reaction system was as shown in Table 1.
TABLE 1 reaction System configuration Table
Component (A) Volume of
10 XDPCRBuffer (containing Taq enzyme and dNTP) 4.5μL
Upstream and downstream primers 0.5 mu L each
Probe with a probe tip 1μL
Nucleic acid templates 2μL
Nuclease-free water 26.5μL
Total volume of 35μL
6. Droplet preparation and reaction
Fully and uniformly mixing the prepared reaction system, preparing an oil phase and adding the oil phase into an oil phase plate, carefully adding samples into sample adding holes in the digital PCR eight rows, placing the digital PCR eight rows into a PCR full-automatic liquid drop preparation instrument according to marks, transferring the digital PCR eight rows into the PCR amplification instrument, pre-denaturing the digital PCR eight rows for 2min at 95 ℃, denaturing the digital PCR eight rows for 15s at 95 ℃, and annealing the digital PCR eight rows for 30s at 54 ℃ for 45 cycles.
7. Chip reading and analysis
And observing and detecting a fluorescence result by using a PCR biochip reader with analysis software, calculating the copy number of the template in the positive sample, and calculating the total copy number in the sample according to the dilution factor in the reaction system.
8. Data analysis
The positive template detection result shows that the detected positive samples show positive signals, the negative control does not show fluorescent signals, the reaction system in the table 1 needs to be operated on ice in the whole process in a dark place when being configured, the negative control group does not contain the positive sample of the porcine sapelovirus, and the same volume of water without the nuclease is added to fill the volume of the reaction system.
2. Establishment of chip type digital PCR detection method for porcine sapelovirus
The amplification efficiency and reaction system of digital PCR are related to various factors, wherein the annealing temperature and the concentration ratio of primer probes are important parameters in the reaction system, and directly influence the generation number and fluorescence intensity of liquid drops. The test optimizes the annealing temperature and the primer probe concentration ratio so as to establish the optimal annealing temperature and primer probe concentration ratio. The result of PCR amplification by setting five annealing temperatures of 52 ℃,54 ℃, 56 ℃, 58 ℃ and 60 ℃ on a digital PCR amplification instrument proves that the optimal annealing temperature of the chip type digital PCR method of the porcine sapelovirus is 54 ℃. The concentration ratio of the primer probe, namely the concentration ratio of the upstream (or downstream) primer to the probe, is respectively set to 200nM/1000nM, 500nM/1000nM, 800nM/1000nM, 500nM/1500nM, 500nM/1200nM and 500nM/800nM, and the reaction system is configured, wherein when the annealing temperature is 54 ℃, the concentration ratio of the primer probe is 500nM/1500nM, and the result is as shown in figure 2, the interval between the fluorescence values of positive liquid drops and negative liquid drops is the largest, the positive liquid drops are the most concentrated, the quantity of intermediate impurity liquid drops is the least, the amplification effect is the best, and finally, the optimal annealing temperature of the chip type digital PCR of the porcine sapelovirus is established to be 54 ℃ and the optimal concentration ratio of the primer probe is 500nM/1500nM.
The optimal annealing temperature (i.e., 54 ℃) and the optimal primer probe concentration ratio (i.e., 500nM/1500 nM) determined in this section are used in the following experiments.
3. Specificity test of chip type digital PCR (polymerase chain reaction) detection method for porcine sapelovirus
To establish the specificity of the chip-type digital PCR detection method of the porcine sapelovirus according to the invention, the common diarrhea/reproductive disorder pathogens of farms are used: the method comprises the steps of taking porcine delta coronavirus (PDCoV), porcine Epidemic Diarrhea Virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine Circovirus (PCV) and pseudorabies virus (PRV) nucleic acid as specific samples, simultaneously taking nuclease-free water as negative control, carrying out digital PCR amplification under the determined reaction conditions, and detecting only porcine sapelovirus by the established chip type digital PCR, wherein obvious FAM channel droplet concentrated distribution appears, and other parts are negative, so that the detection method is proved to have good specificity.
4. Sensitivity test of chip type digital PCR (polymerase chain reaction) detection method for porcine sapelovirus
In order to explore the minimum detection limit of the chip type digital PCR detection method of the porcine sapelovirus established by the invention, the constructed porcine sapelovirus standard plasmid (concentration is 3.12 multiplied by 10 9 The copies/. Mu.L) are subjected to gradient dilution according to a ratio of 10 times, respectively used as templates for digital PCR amplification, each gradient concentration is repeated for 3 times, nuclease-free water is used as a negative control, the sensitivity result of the established chip type digital PCR detection method for the porcine sapelovirus is shown as a graph in fig. 4, and the result shows that the sensitivity of the method to the porcine sapelovirus can reach a single copy (3.12 copies/. Mu.L), and the detection method is higher.
5. Chip type digital PCR (polymerase chain reaction) detection clinical sample application of porcine sapelovirus
The detection of 48 pig diarrhea samples collected from pig farms in Henan, hebei and Shanxi was performed by using a cdPCR and TaqMan real-time fluorescent quantitative PCR (qPCR) method (the results are shown in Table 2), and the infection condition of the pig sapelovirus was analyzed. The analysis of the digital PCR detection results shows that 11 cases of porcine sapelo virus infection in 48 porcine diarrhea samples, 5 cases of porcine sapelo virus infection in 48 porcine diarrhea samples, and the experimental results show that the sensitivity of detecting the porcine sapelo virus by the cdPCR is higher, and the method has more advantages in detecting samples with low viral load. Therefore, the method can be applied to the detection of pig salmeterol virus clinical infection samples in pig farms, and can also realize the detection of early virus infection by virtue of the high sensitivity.
TABLE 2 comparison of clinical samples cdPCR and qPCR
Note that: "-" indicates Negative, no amplification curve was obtained for the sample.
6. Clinical monitoring application of chip type digital PCR (polymerase chain reaction) detection of viral tissue tropism and aerosol of porcine sapelovirus
Many scientific difficulties of viruses need to be studied and solved in an intensive manner depending on the tropism of viral tissues. To study the tropism of swine sapelo virus infected piglets in tissues, experimental piglets were equally divided into challenge groups and blank control groups. 3 piglets of 5 days old are raised in an incubator, and are inoculated with a porcine sapelovirus virus liquid (10) 9 TCID 50 ) After 12 hours, the piglets show obvious diarrhea symptoms. And (3) carrying out virus attack for 24 hours, carrying out dissecting examination to obtain tissue organ samples such as heart, liver, brain, spleen, stomach, lung, mesenteric lymph node, inguinal lymph node, subiliac lymph node and the like, storing at-80 ℃, then carrying out RNA extraction operation, and obtaining the tissue organ copy number of the porcine sapelovirus by using a cd-PCR detection method of the porcine sapelovirus. The test results are shown in Table 3, and the analysis shows that the porcine sapelovirus has multi-tissue tropism, the virus can be detected at the heart, liver, brain, lung and other parts, and the virus content in the mesenteric lymph nodeHigher.
TABLE 3 study of tissue phagocytosis of porcine sapelo virus
With the development of modern intensive breeding industry and the continuous increase of breeding density, pathogens transmitted in the air pose a threat to animal health and cause great economic loss to pig industry. In order to prevent the pig sapelo virus from bringing harm to pig industry from the aspects of biological safety and aerosol, the transmission condition of the pig sapelo virus in the air is deeply explored, and the method has important theoretical significance for formulating practical and effective prevention and control measures. The chip type digital PCR detection method for the porcine sapelovirus has higher sensitivity and accuracy, can realize absolute quantification of micro-virus particles, and provides basic data and technical support for early warning and prevention of virus propagation through air. In this experiment, 3-5 day-old piglets were challenged with porcine sapelo virus (10) 9 TCID 50 ) The piglets then had symptoms such as diarrhea, and the symptoms of diarrhea were most pronounced in the piglets at 72 hours. Air samples were collected at horizontal distances of 0m, 2m and 4m from the vaccinated piglet group using a langerhans air aerosol collector at 24 hours and 72 hours after challenge, respectively. After RNA in the sampling solution is extracted, the copy number of the porcine sapelovirus is detected by using the established digital PCR detection method, and the result is shown in figure 5. Studies have demonstrated that porcine sapelo virus can be transmitted through aerosols, and the results indicate that the closer the content of porcine sapelo virus particles in the aerosols is to the vaccinated piglets, the more the content is. At 72 hours after the challenge, the virus particles above the challenge piglet group (horizontal distance of 0 m) can reach 49444copies/m 3 . The swine sapelovirus chip type digital PCR detection method established by the invention is utilized to carry out the research on the propagation rule of swine sapelovirus aerosol for the first time, and the research result provides an effective data basis for the research on the propagation rule of swine sapelovirus in air. The virus aerosol is extremely harmful to public health and animal health, and the invention is specific to the porcine sapelo virusAn understanding of the role in aerosol disease transmission deserves urgent attention.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (7)

1. A primer pair and probe combination for detecting a porcine sapelo virus strain PSV_BRA/UEL-WB20/13 by chip type digital PCR is characterized in that the primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. The primer pair and probe combination of claim 1, wherein the probe is linked at its 5 'end to a FAM group and at its 3' end to a BHQ1 group.
3. Use of a primer pair and probe combination according to claim 1 or 2 for the preparation of a chip-type digital PCR kit for detecting the porcine sapelo strain psv_bral/UEL-WB 20/13.
4. A chip-type digital PCR kit for detecting porcine sapelo strain psv_bral/UEL-WB 20/13, comprising the primer pair and probe combination of claim 1 or 2.
5. The digital PCR chip kit of claim 4, wherein the concentration ratio of the primer pair and the probe is 500nM/1500nM.
6. The chip-type digital PCR kit as set forth in claim 4, wherein the chip-type digital PCR kit further includes a digital PCR Buffer, a salpeter virus positive detection template, and nuclease-free water.
7. The chip-type digital PCR kit as set forth in claim 4, wherein the reaction procedure of the chip-type digital PCR kit is as follows: pre-denaturation at 95℃for 2min, denaturation at 95℃for 15s, annealing at 54℃for 30s,45 cycles.
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Porcine teschovirus, sapelovirus, and enterovirus in Swiss pigs: multiplex RT-PCR investigation of viral frequencies and disease association;Tamara Stäubli等;《Journal of Veterinary Diagnostic Investigation》;第33卷(第5期);摘要，表1 *
李莉，胡志东.《临床检验仪器》.北京：中国医药科技出版社,2019,第277-278页. *
猪萨佩罗病毒实时荧光定量RT-PCR检测方法的建立与应用;郭博;方和俊;刘小琬;杨凡;刘鹏娟;黄剑波;李萍;徐志文;朱玲;;中国兽医科学(第09期);摘要，第1.3节 *

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