CN115725788A

CN115725788A - Primer and TaqMan probe for detecting feline parvovirus and application thereof

Info

Publication number: CN115725788A
Application number: CN202211083435.8A
Authority: CN
Inventors: 李淑艳; 王勇; 穆苑苑; 刘璇; 霍欣蕊; 吴海强; 谢祥宇; 王润
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-09-06
Filing date: 2022-09-06
Publication date: 2023-03-03

Abstract

The invention discloses a primer and a TaqMan probe for detecting feline parvovirus and application thereof, belonging to the field of molecular biology. The primers comprise an upstream detection primer shown as SEQ ID NO:1, and the downstream detection primer is shown as SEQ ID NO:2 is shown in the specification; the fluorescent probe is shown as SEQ ID NO:3, respectively. The invention utilizes the primers and the probes to detect the feline parvovirus, and uses the fluorescence signal intensity which changes along with the amplification of the target gene fragment to quickly detect and identify FeCHPV, overcomes the defects of the conventional PCR detection, has the characteristics of high efficiency, accuracy, specificity, repeatability, capability of quantitative detection and the like, and provides an effective technical means for the detection of the feline parvovirus.

Description

Primer and TaqMan probe for detecting feline parvovirus and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a primer and a TaqMan probe for detecting feline parvovirus and application thereof.

Background

Feline Parvovirus (FPV), a single-stranded DNA virus that causes feline panleukopenia, is a highly contagious and often fatal disease of cats and other felines. In 2019, a novel FPV, the cat charpy virus (FeChPV), was found in fecal samples from cats housed in a multi-facility feline, british columbia, department during outbreak of Acute Gastroenteritis (AGE). FeCHPV belongs to the family feline parvoviridae (subfamily hamapvorinae) and belongs to the genus chaphamapvorinae, is a small-sized, non-enveloped icosahedral virus, and has single-stranded linear DNA and a genome length of 4.0-6.0kb. Has 3 Open Reading Frames (ORF) respectively encoding non-structural proteins NS1 (ORF 1), nucleoprotein NP (ORF 3) and major capsid protein VP (ORF 2), wherein NS1 encodes 658 amino acids (aa), NP encodes 508 amino acids (aa) and VP encodes 187 amino acids (aa). FeChPV infects vertebrates such as dogs, cats, bats, rats, mice, monkeys, pigs, birds, turkeys, peacocks, chickens, and badgers, and is the most common pathogen in the intestinal tract of cats and is highly prevalent.

At present, feCHPV is a new virus which can cause diarrhea, abdominal pain, fever and the like of cats. However, the clinical studies on feline viruses are mostly Feline Parvovirus (FPV), feline Rotavirus (FRV), feline coronavirus (FCoV), feline Calicivirus (FCV), etc., and the studies on FeChPV are rare.

Some common virus detection methods exist clinically, the conventional Polymerase Chain Reaction (PCR) has complex operation and low sensitivity, and the result is only qualitative; the result of the real-time fluorescent quantitative PCR detection method based on SYBR Green I is easy to generate false positive; the loop-mediated isothermal amplification (LAMP) is easy to cause aerosol pollution, so that a clinical detection method which has the advantages of rapidness, high efficiency, good specificity and reproducibility, visual reaction results and the like is established, and the method has important significance for clinically monitoring, preventing and controlling the diarrhea of the cats.

Disclosure of Invention

The invention aims to provide a primer and a TaqMan probe for detecting feline parvovirus and application thereof, and aims to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a primer and a fluorescent probe for detecting feline parvovirus, wherein the primer comprises an upstream detection primer and a downstream detection primer, and the nucleotide sequence of the primer is shown as SEQ ID NO: 1-2; the nucleotide sequence of the fluorescent probe is shown as SEQ ID NO:3, respectively.

Preferably, the feline parvovirus is feline charpy virus.

Preferably, the reporter fluorophore at the 5 'end of the fluorescent probe is FAM, and the quencher fluorophore at the 3' end of the fluorescent probe is BHQ1.

The invention also provides a kit for detecting the feline parvovirus, which comprises the primer and the fluorescent probe.

The present invention also provides a method for detecting feline parvovirus for non-disease diagnostic purposes, comprising: and (3) carrying out real-time fluorescent quantitative reaction by using the primer and the fluorescent probe, after the reaction is finished, comparing the Ct value of the template DNA with a standard curve to obtain the copy concentration of the target gene fragment of the feline parvovirus in the template DNA, and judging whether the feline parvovirus exists or not based on the obtained copy concentration.

Preferably, the reaction system for performing the real-time fluorescent quantitative reaction using the primer and the fluorescent probe comprises: 2 × TaqMan Fast qPCR premix (Low ROX) 10 μ L, ddH ₂ O8 muL, upstream primer 0.4 muL, downstream primer 0.4 muL, template 1 muL and 0.2 muL fluorescent probe; reaction procedure: 180s at 94 ℃, 5s at 94 ℃, 30s at 60 ℃ and 40 cycles.

The invention also provides application of the primer and the fluorescent probe in preparation of a product for detecting the feline parvovirus.

Preferably, the product comprises a medicament, a reagent or a kit.

The invention discloses the following technical effects:

the invention adopts a pair of specific primers and a TaqMan probe, the TaqMan probe marked with fluorescein is mixed with template DNA, the thermal cycle of high-temperature denaturation, low-temperature renaturation and proper-temperature extension is completed, the polymerase chain reaction rule is followed, the TaqMan probe which is complementarily matched with the template DNA is cut off, the fluorescein is dissociated in a reaction system, fluorescence is emitted under specific laser, the amplified target gene segment is exponentially increased along with the increase of the cycle number, and FeCHPV is rapidly detected and identified by using the corresponding fluorescence signal intensity which is changed along with the amplification through real-time detection. The invention not only overcomes the error in the conventional PCR method, but also has strong sensitivity, high specificity, good repeatability, simple operation, short detection time and high accuracy. The minimum detection sample size is 3.75 × 10 ¹ Individual DNA copies/. Mu.L, are 10-100 times more sensitive than conventional PCR methods. The invention is a universal detection method, can carry out qualitative and quantitative experiments on a large number of samples in a PCR detection instrument, and can be used for clinically, rapidly, efficiently and specifically monitoring FeCHPV.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a FeCHPV standard curve;

FIG. 2 shows the result of the specificity test of FeCHPV;

FIG. 3 shows the results of a FeCHPV sensitivity test;

FIG. 4 shows the result of the repeatability test of FeCHPV;

FIG. 5 shows the accuracy test results of FeCHPV, in which M represents Marker, N is negative control, 1-8 represents copy number of 3.75X 10 ⁸ copies/. Mu.L to 3.75X 10 ¹ copies/. Mu.L of standard plasmid.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description 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. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 effectiveness test of real-time fluorescent quantitative PCR detection method of FeCHPV

1. Primer and TaqMan probe design

FeCHPV used in the experiment is detected from a positive sample of domestic cat viral diarrhea feces, is subjected to sequencing identification by a company, is uploaded to GenBank (GenBank number: MT 708231.1), is subjected to gene sequence comparison by using DNAstar software Megalign, is subjected to primer and probe design in a VP gene conserved region of FeCHPV, and is synthesized by general biology (Anhui) GmbH.

An upstream detection primer: 5 'CACCTGATTCTACTGGGGCACAA-3' (SEQ ID NO: 1);

downstream detection primers: 5 'GTCATTGAATTTTTTTTTCCTGGTCTT-3' (SEQ ID NO: 2);

a fluorescent probe: 5 'ATGATGCCGACTGGAGTTTTTTGGGAC-3' (SEQ ID NO: 3).

2. Extraction of viral nucleic acids

The virus genome is extracted from the collected samples according to the instructions of a Tiangen genome kit (Tiangen, beijing), and then the virus genome is stored at the temperature of 20 ℃ below zero for later use.

3. Construction of recombinant plasmids

3.1 connection

The purified DNA fragment of interest was ligated into the pMD-19T vector using the TA cloning method. The ligation conditions were 16 ℃ for ligation for 3h. The linking system is as follows: 4 mu L of purified product, 1 mu L of pMD-19T (Baoriyao, beijing) and 5 mu L of Solution I (Baoriyao, beijing).

3.2 transformation

The ligation product was then transformed into DH5a competent cells by the following specific procedures:

(1) Taking out DH5 alpha competent cells in advance, and placing the cells on ice to fully melt the cells;

(2) Add ligation product 5. Mu.L to 50. Mu.L of DH 5. Alpha. Competent cells;

(3) Incubating at 4 ℃ for 30min, then carrying out water bath at 42 ℃ for 90s, and then quickly transferring ice to carry out ice bath for 150s;

(4) Adding 600 μ L of nonresistant LB medium preheated to 37 deg.C, and culturing at 37 deg.C with shaking table at 200rpm for 50min;

(5) Centrifuging at 6000rpm for 3min, discarding 500 μ L of supernatant, and reserving 100 μ L of liquid to fully resuspend bacterial precipitation;

(6) The resuspended liquid was aseptically spread on ampicillin-resistant LB solid medium and cultured at 37 ℃ for 8 hours.

3.3 plasmid extraction and sequencing

After overnight culture, single positive colonies in solid medium were picked and cultured in 5mL of ampicillin-resistant LB liquid medium at 37 ℃ for 12h with a shaker at 200 rpm. Meanwhile, PCR of bacteria liquid is carried out for verification, and plasmid extraction is carried out. The detailed steps for extracting plasmids are as follows:

(1) Column equilibration step: add 500. Mu.L of equilibration fluid BL to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste:

(2) Adding the bacterial liquid into a centrifugal tube, centrifuging at 12000rpm for 1min, and completely removing the supernatant;

(3) Adding 250 mu LBuffer P1 into the sediment, and carrying out vortex oscillation to completely resuspend the sediment;

(4) Adding 250 mu LBuffer P2 into the suspension in the previous step, and turning up and down for 6-8 times;

(5) Adding 350 mu LBuffer P3 into the suspension in the previous step, turning the suspension up and down for 6 to 8 times, and centrifuging the suspension at 12000rpm for 10min;

(6) Sucking out the supernatant to a filter column CS, and centrifuging at 12000rpm for 2min;

(7) Adding the centrifuged liquid into an adsorption column which finishes the column balancing step, centrifuging at 12000rpm for 45s, and discarding the waste liquid;

(8) Adding 500 mu L of buffer solution PD into the adsorption column, centrifuging at 12000rpm for 45s, and discarding the waste liquid;

(9) Adding 600 μ L of rinsing solution PW into the adsorption column, centrifuging at 12000rpm for 45s, and discarding the waste solution;

(10) Repeating the step (9);

(11) Adding no liquid into the adsorption column, centrifuging at 12000rpm for 2min, and completely removing the liquid in the adsorption column;

(12) Transferring the adsorption column into a new 1.5mL centrifuge tube, dripping 50 μ L of eluent TB into the middle part of the adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min;

(13) The obtained recombinant plasmid is put into a temperature of minus 20 ℃ for storage and standby, and meanwhile, part of the plasmid is extracted and sent to general biology (Anhui) GmbH for sequencing verification.

Example 2 real-time fluorescent quantitative PCR reaction and Standard Curve preparation

The concentration of the recombinant plasmid was measured using an ND-2000 spectrophotometer, and the result was substituted into the formula: DNA concentration (copy number) = [6.02 × 10 ²³ Copy/mol plasmid concentration (ng/. Mu.L). Times.10 ^-9 ]/[ DNA length in nucleotide units X660 g/mol]Calculating the copy number of FeCHPV recombinant plasmid to be 3.75 multiplied by 10 ⁸ copies/μL。

The recombinant plasmid is diluted by ten-fold gradient to obtain the copy number of 3.75 multiplied by 10 ⁸ copies/. Mu.L to 3.75X 10 ¹ copies/. Mu.L of standard plasmid. The qPCR assay was performed on the LightCycle96 assay System (Roche, sweden). The reaction system comprises: 2 × TaqMan Fast qPCR premix (Low ROX) 10 μ L, ddH ₂ O8. Mu.L, forward primer 0.4. Mu.L, reverse primer 0.4. Mu.L, template 1. Mu.L, 0.2. Mu.L probe. Reaction procedures are as follows: 180s at 94 ℃, 5s at 94 ℃ and 30s at 60 ℃ for 40 cycles.

A standard curve for establishing TaqMan real-time fluorescent quantitative PCR of FeCHPV is shown in figure 1, the abscissa represents the logarithm of the plasmid copy number, and the ordinate is a Ct value. The standard curve regression equation is: y = -3.3786X +39.718, correlation coefficient R ² 0.9995, and the amplification efficiency was 98%. The method shows that the target gene amplification and Ct values with different concentrations have good linear relation, so that the reaction condition and the standard curve can be used for quantitative analysis of corresponding genes.

Example 3 real-time fluorescent quantitative PCR reaction specificity assay

Amplifying by using a positive standard of cat charpy virus (FeCHPV), a positive standard of cat boka type 1 virus (FBoV-1), a positive standard of cat kubu virus (FKoV), a positive standard of cat calicivirus (FCV), a positive standard of cat boka type 3 virus (FBoV-3), a positive standard of cat parvovirus (FPV), a positive sample of cat herpes virus (FHV, felo-vax PCT cat vaccine, boringer's group) as a template, establishing a fluorescent quantitative PCR reaction,by ddH ₂ O is a negative control.

As shown in FIG. 2, only FeCHPV showed a specific amplification curve in the LightCycle96 detection system, and no specific amplification occurred in any other virus samples, indicating that the method has good specificity.

Example 4 real-time fluorescent quantitative PCR reaction sensitivity test

The copy number obtained in example 2 was 3.75X 10 ⁸ copies/. Mu.L to 3.75X 10 ¹ The amplification curve obtained by real-time fluorescent quantitative PCR amplification with the standard plasmid of copies/mu L as the template is shown in FIG. 3, and the result shows that the lowest detection line of the method of the invention is 3.75 multiplied by 10 ¹ copies/. Mu.L, which is 10-100 times more sensitive than the conventional PCR method, the results are shown in FIG. 3.

Example 5 real-time fluorescent quantitative PCR reaction repeatability test

The copy number obtained in example 2 was 3.75X 10 ⁸ copies/. Mu.L to 3.75X 10 ¹ Taking the standard plasmids of copies/mu L as templates, carrying out a repetitive experiment, setting 3 repetitive groups for the standard plasmids of each concentration, and carrying out an intra-group repetitive experiment; meanwhile, standard plasmids of each concentration were selected for different days, and different experimenters performed 3 independent intergroup repeatability tests, respectively, and the results are shown in fig. 4. And finally, counting the average value, standard deviation and variation coefficient (%) of the repeatability tests in the groups and the repeatability tests among the groups so as to evaluate the repeatability and stability of the detection method. The variation coefficients in the groups and between the groups are 0.42% -2.39% and 0.39% -2.02% respectively, and the variation coefficients are less than 5%, which shows that the method has good stability and repeatability. The results are shown in Table 1.

TABLE 1FeCHPV TaqMan-based real-time fluorescent quantitation PCR repeatability test

Example 6 real-time fluorescent quantitative PCR reaction accuracy test

The copy number obtained in example 2 was 3.75X 10 ⁸ copies/. Mu.L to 3.75X 10 ¹ The copy/. Mu.L standard plasmid is used as a template, and the PCR method is adopted for detection after the upstream primer and the downstream primer are added.

Reaction system: 2 × Taq PCR MasterMix II (premix enzyme) 5 μ L, ddH ₂ O3. Mu.L, plasmid 1. Mu.L, and upstream and downstream primers 0.5. Mu.L, respectively. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s; annealing at 60 ℃ for 30s; extension at 72 ℃ for 30s; further extension for 10min at 72 ℃; after 35 cycles, images are kept by observing and photographing under an ultraviolet lamp after electrophoresis.

As shown in fig. 5: the DNA is marked as 2000bp, electrophoresis channels 1-8 are added with samples to be diluted by 10 times, and the plasmid dilution range is 3.75 multiplied by 10 ⁸ -3.75×10 ¹ copies/. Mu.L; marker is added to the M electrophoresis track, and the N electric lane is a negative control. The lowest detection limit of PCR was 3.75X 10 ³ copies/. Mu.L. Compared with the TaqMan-based real-time fluorescent quantitative PCR of FeCHPV, the minimum detection limit of the kit is 3.75 multiplied by 10 ¹ The sensitivity of copies/mu L is 10-100 times higher than that of the conventional PCR method, and the concentration of FeCHPV can be quantitatively and accurately detected through establishing a standard curve. It can be seen that the present invention has a higher degree of accuracy.

The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The primers and the fluorescent probe for detecting the feline parvovirus are characterized by comprising an upstream detection primer and a downstream detection primer, wherein the nucleotide sequences of the upstream detection primer and the downstream detection primer are shown as SEQ ID NO: 1-2; the nucleotide sequence of the fluorescent probe is shown as SEQ ID NO:3, respectively.

2. The primer and fluorescent probe of claim 1, wherein the feline parvovirus is feline chalcone virus.

3. The primer and fluorescent probe of claim 1, wherein the reporter fluorophore at the 5 'end of the fluorescent probe is FAM and the quencher fluorophore at the 3' end of the fluorescent probe is BHQ1.

4. A kit for detecting feline parvovirus, comprising the primer of any one of claims 1 to 3 and a fluorescent probe.

5. A method for detecting feline parvovirus for non-disease diagnostic purposes, comprising: the primer and the fluorescent probe of any one of claims 1-3 are used for real-time fluorescent quantitative reaction, after the reaction is finished, the Ct value of the template DNA is compared with a standard curve to obtain the copy concentration of the target gene fragment of the feline parvovirus in the template DNA, and whether the feline parvovirus exists is judged based on the obtained copy concentration.

6. The method of claim 5, wherein the reaction system for performing the real-time fluorescent quantitative reaction using the primer and the fluorescent probe comprises: 2 × TaqMan Fast qPCR premix (Low ROX) 10 μ L, ddH ₂ O8 muL, upstream primer 0.4 muL, downstream primer 0.4 muL, template 1 muL and 0.2 muL fluorescent probe; reaction procedure: 180s at 94 ℃, 5s at 94 ℃ and 30s at 60 ℃ for 40 cycles.

7. Use of the primers and fluorescent probe as defined in any one of claims 1 to 3 for the preparation of a product for detecting feline parvovirus.

8. The use of claim 7, wherein the product comprises a medicament, a reagent or a kit.