Dual fluorescent quantitative PCR detection primer for FHV-1 and FCV and application of dual fluorescent quantitative PCR detection primer

Abstract

The invention discloses a dual fluorescent quantitative PCR detection primer for FHV-1 and FCV and application thereof, the primer pair and probe combination comprises: 1) a primer pair and a probe for detecting cat herpes virus strain FHV, the sequence of the upstream primer is SEQ ID NO: 1, the sequence of the downstream primer is SEQ ID NO: 2, and the sequence of the probe is SEQ ID NO: 3; the primer pair and the probe are used for detecting the FCV of the feline calicivirus strain, the sequence of an upstream primer of the primer pair is SEQ ID NO: 4, the sequence of a downstream primer of the primer pair is SEQ ID NO: 5, and the sequence of the probe is SEQ ID NO: 6; and 3) a primer pair and a probe used as a cat exogenous internal standard for internal reference detection, the sequence of an upstream primer is SEQ ID NO: 7, the sequence of a downstream primer is SEQ ID NO: 8, and the sequence of the probe is SEQ ID NO: 9. According to the present invention, the newly constructed dual Taqman fluorescent quantitative PCR is adopted to perform microencapsulation pretreatment, such that the environmental pollution during the operation process is reduced, the specificity is strong, the sensitivity is high, the repeatability is good, and the technical support is provided for clinical cat respiratory system disease pathogen differential diagnosis and pet health monitoring.

Description

Technical Field

[0001] The invention belongs to the field of gene detection, and specifically relates to a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV and an application thereof. Background Art

[0002] With the continuous development of the pet market, the respiratory problems of companion animals are also increasing rapidly. Among them, FHV-1 and FCV are the most common respiratory infections, with the highest prevalence, complex infection symptoms, and a certain mortality rate, which have serious impacts on the health of companion animals and humans. At present, in the process of clinical diagnosis and treatment, the commonly used detection method is still the colloidal gold test strip. Although this method is convenient, its sensitivity is poor. In particular, both FHV-1 and FCV infections have an incubation period and a virus-carrying stage. In this stage, this method is prone to false negatives. The fluorescence quantitative PCR detection method has the characteristics of high efficiency, sensitivity, and speed. It has been widely used in clinical virus detection and is of great significance for the early diagnosis and prevention of pathogens that infect companion animals. In order to establish a new method for the rapid and convenient diagnosis of feline herpes virus (FHV-1) and feline calicivirus (FCV), a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV and its application are needed. Summary of the invention

[0003] The invention aims to provide a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV and application thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] The primer pair and probe combination described in the present invention includes:

[0006] 1) A primer pair and a probe for detecting a feline herpesvirus strain FHV, wherein the sequence of the upstream primer is SEQ ID NO: 1, the sequence of the downstream primer is SEQ ID NO: 2, and the sequence of the probe is SEQ ID NO: 3;

[0007] 2) A primer pair and a probe for detecting feline calicivirus strain FCV, wherein the sequence of the upstream primer is SEQ ID NO: 4, the sequence of the downstream primer is SEQ ID NO: 5, and the sequence of the probe is SEQ ID NO: 6;

[0008] 3) A primer pair and a probe for detecting a cat exogenous internal standard as an internal reference, wherein the sequence of the upstream primer is SEQ ID NO: 7, the sequence of the downstream primer is SEQ ID NO: 8, and the sequence of the probe is SEQ ID NO: 9.

[0009] A multiplex fluorescence quantitative PCR detection kit comprises the primer pair and probe combination.

[0010] A method for detecting feline herpesvirus and feline calicivirus, wherein the method uses the primer pair and probe combination to perform multiplex PCR fluorescence quantitative detection.

[0011] Further, the method described herein comprises the following steps: the primers for the feline herpesvirus strain are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL; the primers for the feline calicivirus strain are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL; and the primers for the cat exogenous internal standard are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL.

[0012] 5. The method according to claim 3, characterized in that the reaction conditions are 50°C for 15 min for one cycle; 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 40 cycles; 25°C for 10 s for one cycle.

[0013] Compared with the prior art, the present invention adopts the above technical solution, and the biggest feature is:

[0014] The present invention utilizes the newly constructed dual Taqman fluorescent quantitative PCR for microencapsulation pretreatment, which reduces environmental pollution during the operation process, has strong specificity, high sensitivity, and good repeatability, and provides technical support for clinical differential diagnosis of pathogens of feline respiratory diseases and pet health monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The standard curve of FHV-1 of the dual fluorescence quantitative PCR detection primer of FHV-1 and FCV of the present invention

[0016] Figure 2 A standard curve diagram of FCV of a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV of the present invention;

[0017] Figure 3 A standard curve diagram of a cat-derived internal reference IC of a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV of the present invention;

[0018] Figure 4 A graph showing the specificity test results of dual fluorescence quantitative PCR of a dual fluorescence quantitative PCR detection primer for FHV-1 and FCV of the present invention;

[0019] Among them, 1: IC(1)2: IC(2)3: FHV-1(1)4: FHV-1(2)5: FCV(1)6: FCV(2)7-10: FCoV, Mf, IV-A and Rnase free water

[0020] Figure 5 The sensitivity test results of the dual fluorescence quantitative PCR detection primers for FHV-1 and FCV of the present invention - FAM channel detection diagram;

[0021] Among them, 1-6: 1×10 1 copi es / μL~1×10 6 copies / μL of FHV-17-12: 1×

[0022] 10 1 copi es / μL~1×10 6 copies / μL of FCV 13: IC 14: ddH 2 O. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0024] The objective of the present invention is achieved through the following technical solutions:

[0025] FHV-1 and FPV virus-positive samples were provided by the School of Pet Science and Technology of Jiangsu Vocational College of Agriculture and Animal Husbandry Science and Technology; Feline coronavirus (FCoV), Mycoplasma (M.felis), and influenza A virus (IV-A) were provided by Shanghai Veterinary Research Institute; viral genome kits, plasmid extraction kits, etc. were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; 2XPhanta Max Master Mix, TransI ntroEL Transfection reagent, and DH5α competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; pMD19-T vector and DL500DNA Marker were purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0026] Case 1 Design and synthesis of primers and probes

[0027] According to the sequences of FHV-1 (OR504662) GC gene and FCV (OP904215) VP1 gene registered in GenBank, MEGA7.0 software was used to perform homology analysis and align the conserved region, and Primer Premier 5.0 software was used to design primer sequences for FHV-1 and FCV as well as cat exogenous internal standards, i.e., internal references, and the primer sequences used for the probes are shown in Table 1 below. All were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0028] Table 1 Primers and probes sequences

[0029]

[0030] Construction of recombinant plasmid standards

[0031] Select the DNA or cDNA of FHV-1, FCV and cat exogenous internal standard (as internal reference detection, IC) as templates, and use the primers in Table 1 to amplify the corresponding fragments. The cat exogenous internal standard is an artificially synthesized DNA sequence that has no crossover with the FHV-1 and FCV genes. The PCR products were recovered and cloned into the pMD19-T vector respectively to construct positive recombinant plasmids pMD19-TG (FHV-1), pMD19-TP (FCV), and pMD19-TC (internal reference). After PCR identification of the culture solution, the concentration of the recombinant plasmid was determined using a Drop2000 ultra-micro spectrophotometer and sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing. The copy number was calculated according to the concentration results displayed by the computer software, and the formula was: copy number / μL = (plasmid concentration ng / μL × 6.02 × 10 23 ) / (10 9 × genome length × 660Da / bp). 10-fold dilution to 1×10 1 Copies / μL~1×10 5 copies / μL, kept at -20℃ for future use.

[0032] Optimization of reaction conditions and establishment of standard curve

[0033] The recombinant plasmids pMD19-TG, pMD19-TP, and pMD19-TC were diluted in equal proportions and mixed at a volume ratio of 1:1:1 to obtain a dilution of 10 1 -10 5 The plasmid standard mixture was used as a template, and specific primers and Taqman probes were used to amplify in the same system to draw a standard curve. The primer concentration (0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L), probe concentration (0.05μmol / L, 0.1μmol / L, 0.15μmol / L, 0.2μmol / L), annealing temperature (55℃-63℃) and other conditions were optimized according to the square array method to obtain the best reaction conditions for this detection method.

[0034] The recombinant plasmids pMD19-TG, pMD19-TP, and pMD19-TC were diluted to the same concentration in equal proportions, mixed at a volume ratio of 1:1:1, and then the dilution was 101 -10 6 The plasmid standard mixture was used as a template and amplified by fluorescent quantitative PCR according to the optimized reaction conditions. A standard curve was drawn according to the Ct value and gene copy number.

[0035] Specificity test

[0036] The gene sequences of FHV-1, FCV, FCoV, Mf, IV-A and cat-derived internal standard recombinant plasmids were used as templates, the recombinant positive plasmid standards were used as positive controls, and sterile RNase-free water was used as negative controls. The established dual Taqman fluorescence quantitative PCR method was used to evaluate its specificity and the effect of the cat-derived internal standard on the overall reaction.

[0037] Sensitivity test

[0038] The recombinant plasmid standards pMD19-TG and pMD19-TP were diluted to the same concentration in equal proportions, mixed at a volume ratio of 1:1, and then the dilution ratio was 10 1 -10 6 As a template, it was amplified according to the newly established method to evaluate its sensitivity.

[0039] Repeatability test

[0040] The recombinant plasmid standards pMD19-TG and pMD19-TP were diluted to the same concentration in equal proportions, mixed at a volume ratio of 1:1, and then the dilution ratio was 10 1 , 10 3 , 10 5 The positive standard mixture was used as a template, and the newly developed detection method was used to detect the amplification results of the same sample, and the intra-group repeat test was performed; using the standard sample with the same dilution as a template, amplification was performed at different times to complete three inter-group repeatability tests, and the corresponding coefficient of variation was calculated.

[0041] Testing of clinical samples

[0042] 33 samples of eye, mouth and nose secretions from clinically ill cats were vortexed in the premix for 15 seconds and centrifuged at 5000r / min for 5 minutes, and the supernatant was transferred to a new sterile 1.5ml centrifuge tube. The dual Taqman fluorescent quantitative PCR established by the present invention and the conventional single PCR and RT-PCR in the literature were used for detection. The positive coincidence rate, negative coincidence rate and total coincidence rate were calculated according to the test results. The calculation method is shown in Table 2.

[0043] Table 2 Calculation of compliance rate of sample test results

[0044]

[0045]

[0046] Case 2 Construction and identification results of recombinant plasmid standards

[0047] The recombinant plasmid standards pMD19-TG, pMD19-TP, and pMD19-TC were used as templates for PCR amplification and sequencing. The sequencing results after PCR amplification showed that the sequences of the target genes were correct. The concentrations of FHV-1, FCV, and cat-derived internal reference recombinant plasmids were determined by Drop2000 ultra-micro spectrophotometer to be 3.32×10 3 copies / μL, 3.30×10 4 copies / μL, 3.28×10 4 copies / μL.

[0048] The reaction conditions were optimized according to the matrix method, and the optimal reaction condition system of dual fluorescence quantitative PCR was determined, as shown in Table 3. The optimal reaction conditions were: 50°C for 15 min for 1 cycle; 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 40 cycles; 25°C for 10 s for 1 cycle.

[0049] Table 3 Optimal reaction system

[0050]

[0051]

[0052]

[0053] The results of constructing the standard curve of the detection standard showed that the FAM channel standard curve equation of pMD19-TG was Y=-3.09819x+37.94804, the correlation coefficient R2 was -0.99826, and the amplification efficiency was 1.10266 ( Figure 1 ); The ROX channel standard curve equation of pMD19-TP is Y=-2.87937x+39.20831, the correlation coefficient R2 is -0.99870, and the amplification efficiency is 1.22484 ( Figure 2 ); The HEX channel standard curve equation of pMD19-TC is Y=-3.50566x+34.57185, the correlation coefficient R2 is -0.99900, and the amplification efficiency is 0.92865 ( Figure 3 ).

[0054] Specificity test results

[0055] The gene sequences of FHV-1, FCV, FCoV, Mf, IV-A, and IC (recombinant plasmid of cat internal standard) were used as templates, the recombinant positive plasmid standard was used as the positive control, and sterilized RNase-free water was used as the negative control. The method established by the present invention was used for detection, and two sets of repeated tests were performed. The results showed that only FCV and FHV-1 had amplification curves, while other pathogens, negative controls, and cat internal standards had no amplification curves ( Figure 4 ), indicating that the established method has strong specificity.

[0056] Sensitivity test results

[0057] The standard positive plasmids pMD19-TG and pMD19-T-P were diluted 10 times in different concentration gradients, that is, 1×10 1 Copies / μL~1×10 6 The method established by the present invention was used to detect and evaluate the sensitivity of the method. The results showed that the sensitivity of the method to the two standard plasmids was 1×10 1 copies / μL( Figure 5 ). This indicates that the new method has higher sensitivity.

[0058] Repeatability test

[0059] Take 3 dilutions of 10 1 , 10 3 , 10 5 The pMD19-TG and pMD19-TP mixture of equal volumes was used as templates, and three replicates were set in each group. The newly created method was used for amplification to evaluate the intra-group and inter-group repeatability of the method. The results showed that the coefficient of variation of the intra-group repeatability test was 0.41%-1.66%, and the coefficient of variation of the inter-group repeatability test was 0.76%-1.59%, both less than 2% (Table 4), indicating that the detection method established by the present invention has good repeatability.

[0060] Table 4 Repeatability test results of dual fluorescence PCR

[0061]

[0062]

[0063] Test results of clinical samples

[0064] The method established by the present invention and conventional single PCR / RT-PCR were used to simultaneously detect 33 samples of eye, mouth and nose secretions of infected cats. The results showed that the newly established method detected 12 FHV-1 positive samples, accounting for 36%, and 19 FCV positive samples, accounting for 57.6%, of which 6 samples were mixed with FHV-1 and FCV, accounting for 18.2%. Conventional single PCR detected 11 FHV-1 positive samples, accounting for 23.4%, and 17 FCV positive samples, accounting for 51.6%, of which 5 samples were mixed with FHV-1 and FCV, accounting for 15.2% (Table 5). The positive rate of FHV-1 and FCV was 100%. The total conformity rate of FHV-1 test results was 97.0% (Table 6), and the total conformity rate of FCV test results was 93.95% (Table 7). The sensitivity of the newly established method is higher than that of conventional PCR detection.

[0065] Table 5 Comparison of clinical sample results of two detection methods

[0066]

[0067]

[0068] Table 6 Results of the compliance rate of clinical samples detected by two methods-1

[0069]

[0070]

[0071] Table 7 Results of the compliance rate of clinical samples detected by two methods-2

[0072]

[0073]

[0074] According to the genome sequences of the currently popular FHV-1 and FCV strains, the present invention designs primers and probe primers through comparative analysis, and designs a conservative DNA sequence as an internal reference and internal reference primer. In particular, it has been found in the research process of FCV in recent years that it has extremely strong variability and highly pathogenic strains have appeared, so it is necessary to carefully refer to the genome sequence of the virus. At the same time, when designing primers, probes and internal reference sequences, it is necessary to consider the selection of similar annealing temperatures, dimers and other issues. The addition of internal references greatly increases the detection of the reaction process, and the normal progress of the entire reaction process can be detected in real time throughout the entire process. Through the recombinant construction of plasmid pMD19-T, the selection of internal references, the testing of fluorescent reporter genes, and the optimization of reaction conditions and reaction systems, a dual Taqman fluorescent quantitative PCR detection method was preliminarily constructed. The results of the specificity test showed that only FCV and FHV-1 were detected as positive, and the rest of FCoV, Mf, and IV-A were negative, indicating that the method has strong specificity; the results of the sensitivity test showed that the detection limit of the new method for the virus was 1×10 1 copies / μL, which is consistent with the previously reported detection limit of conventional PCR / RT-PCR for FHV-1 and FCV of 1×10 1 -5×10 1 The copies / μL were comparable, which showed that the sensitivity of the dual Taqman fluorescence quantitative PCR detection method established in this experiment was consistent with that of the fluorescence quantitative method reported in the literature;

[0075] In the comparison of the two methods, it is obvious that the overall detection time of the dual Taqman fluorescence quantitative PCR detection method is much shorter than that of conventional PCR. Since the present invention adds the monitoring of cat exogenous internal standards, it can be equipped with a sensitive and stable portable four-channel fluorescence PCR instrument to achieve rapid and accurate on-site differential diagnosis and result reading. Compared with dual-channel fluorescence PCR, different pathogens can be detected at the same time, improving the timeliness of detection and shortening the time of disease diagnosis. At the same time, by adding internal references before extraction, the whole process of nucleic acid detection can be monitored, including sampling, extraction, amplification and other links. And by detecting whether the internal standard nucleic acid is "starting", it is judged whether there is a probability of false negative caused by "sampling, extraction, and amplification".

[0076] This experiment successfully constructed a dual-probe PCR detection method for FHV-1 and FCV in cats. This method has excellent specificity and high sensitivity. It is easy to operate and has good stability. It can quickly distinguish between FHV-1 and FCV infections in cats, and provide technical support for the differential diagnosis of clinical feline respiratory disease pathogens and pet health monitoring.

[0077] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A dual fluorescence quantitative PCR detection primer pair and probe combination for detecting feline herpes virus and feline calicivirus, characterized in that: The primer pair and probe combination includes: 1) A primer pair and a probe for detecting a feline herpesvirus strain FHV, wherein the sequence of the upstream primer is SEQ ID NO: 1, the sequence of the downstream primer is SEQ ID NO: 2, and the sequence of the probe is SEQ ID NO: 3; 2) A primer pair and a probe for detecting feline calicivirus strain FCV, wherein the sequence of the upstream primer is SEQ ID NO: 4, the sequence of the downstream primer is SEQ ID NO: 5, and the sequence of the probe is SEQ ID NO: 6; 3) A primer pair and a probe for detecting a cat exogenous internal standard as an internal reference, wherein the sequence of the upstream primer is SEQ ID NO: 7, the sequence of the downstream primer is SEQ ID NO: 8, and the sequence of the probe is SEQ ID NO:

9.

2. A multiplex fluorescence quantitative PCR detection kit, characterized in that: The kit comprises the primer pair and probe combination according to claim 1.

3. A method for detecting feline herpesvirus and feline calicivirus, characterized in that, The method is to use the primer pair and probe combination of claim 1 to perform multiplex PCR fluorescence quantitative detection.

4. The method according to claim 3, characterized in that The method comprises the following steps: the primers for the feline herpesvirus strain are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL; the primers for the feline calicivirus strain are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL; and the primers for the cat exogenous internal standard are 0.1 μmol / 20 μL upstream and downstream, and the probe is 0.05 μmol / 20 μL.

5. The method according to claim 3, characterized in that The reaction conditions were 50°C for 15 min for 1 cycle; 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 40 cycles; and 25°C for 10 s for 1 cycle.

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