A feline herpesvirus detection primer set, an isothermal detection kit and application thereof

Abstract

The application provides a feline herpesvirus detection primer group, an isothermal detection kit and application thereof, and belongs to the technical field of virus detection. The application first determines a conservative region from a feline herpesvirus gene sequence, then excludes a region with a complex higher structure in the conservative region, and finally determines a region suitable for primer design. After screening of primer pairs, the primer group screened can accurately and rapidly detect feline herpesvirus, and has high detection repeatability, specificity and detection sensitivity. The application also provides a feline herpesvirus detection method for non-diagnostic purposes. A loop-mediated isothermal amplification reaction is carried out by using the primer group, compared with a detection method using a general PCR detection kit, the reaction can be carried out at a constant temperature, a temperature change is not needed, complex instruments are not needed, the reaction time is short, the result is visualized, and the method is suitable for on-site rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to a feline herpesvirus detection primer set, its isothermal detection kit, and its application. Background Technology

[0002] Feline herpesvirus (FHV), also known as feline rhinotracheitis virus, belongs to the Herpesviridae family and is an enveloped double-stranded DNA virus. The virus is approximately 128 nm to 168 nm in diameter and can readily multiply and passage in feline embryonic kidney, lung, and testicular cell cultures. This virus causes feline rhinotracheitis, and cats are the only natural host for this disease. Infected cats are the primary source of infection, and transmission occurs through direct contact. It mainly affects kittens. In severe cases, infected cats exhibit fever and obvious upper respiratory tract infection. The morbidity rate of feline rhinotracheitis can reach 100%, and the mortality rate can reach 50%. The virus multiplies in the nose, pharynx, trachea, bronchi, tongue, and conjunctiva of infected cats. Given the wide range of lesions caused by this disease, establishing a rapid, accurate, specific, and sensitive detection method is essential.

[0003] Cats tolerate anesthesia and general surgery well and maintain normal blood pressure during surgery. As a laboratory animal specified in the Chinese Pharmacopoeia for testing antihypertensive substances in pharmaceuticals, cats are increasingly used in research on visual deprivation amblyopia and esophageal diseases. Although the use of cats as laboratory animals is becoming more widespread, there are currently no standardized regulations for testing cats in China. While Jiang Hong et al. established a serological detection method for feline rhinotracheitis virus (FHV) as early as 1999, and Liu Baoshan, Lin Ying, and Qu Zhe successively established a PCR detection method for FHV-1 before 2011, neither method has been widely adopted, and there are still no standardized regulations for laboratory cats. Currently, laboratory cats are mostly purchased from vendors or large-scale breeding farms, resulting in mixed origins and unclear genetic, age, microbial, and parasitic carriage status, leading to poor experimental reproducibility and the influence of unknown factors on the results. FHV-1 is one of the most important known feline respiratory diseases, with high morbidity and mortality rates, seriously endangering the health of cats. Currently, there are no institutions in China that conduct clinical testing for feline herpesvirus and other feline susceptible viruses, and there are no reports of testing for FHV-1 in experimental cats. There are also no related test kits available for sale in China. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a rapid and accurate primer set for feline herpesvirus detection, as well as its isothermal detection kit and applications.

[0005] This invention provides a primer set for feline herpesvirus detection, including primer pair FHV-1-F3 / FHV-1-B3, primer pair FHV-1-FIP / FHV-1-BIP, and primer pair FHV-1-LF / FHV-1-LB;

[0006] The nucleotide sequence of the FHV-1-F3 is shown in SEQ ID NO:1;

[0007] The nucleotide sequence of the FHV-1-B3 is shown in SEQ ID NO:2;

[0008] The nucleotide sequence of the FHV-1-FIP is shown in SEQ ID NO:3;

[0009] The nucleotide sequence of the FHV-1-BIP is shown in SEQ ID NO:4;

[0010] The nucleotide sequence of the FHV-1-LF is shown in SEQ ID NO:5;

[0011] The nucleotide sequence of the FHV-1-LB is shown in SEQ ID NO:6.

[0012] This invention provides a feline herpesvirus detection kit, comprising the feline herpesvirus detection primer set.

[0013] Preferably, it also includes a reaction premix;

[0014] The reaction premix contains 0.67 μL Bst 2.0 WarmStart DNA polymerase and 8.33 μL buffer; the reaction premix also contains an acid-base indicator.

[0015] Preferably, the acid-base indicator includes phenol red.

[0016] Preferably, it also includes sample lysis buffer.

[0017] This invention provides the application of the aforementioned feline herpesvirus detection primer set in the preparation of feline herpesvirus detection reagents or kits.

[0018] This invention provides the application of the feline herpesvirus detection primer set or the kit in the detection of feline herpesvirus for non-diagnostic purposes.

[0019] This invention provides a non-diagnostic method for detecting feline herpesvirus loop-mediated isothermal amplification, comprising the following steps:

[0020] 1) The sample to be tested is pyrolyzed to obtain the pyrolyzed sample solution;

[0021] 2) The lysed sample solution from step 1) is subjected to loop-mediated isothermal amplification under the action of the primer set and the reaction premix in the kit to obtain the amplification product;

[0022] 3) Determine whether the sample to be tested contains feline herpesvirus based on the color of the amplification product described in step 2).

[0023] Preferably, in step 1), the solution used for lysis is the sample lysis buffer; the volume ratio of the sample to be tested to the sample lysis buffer is 2:5.

[0024] The pyrolysis conditions were 95°C for 5 minutes.

[0025] Preferably, the reaction system for the loop-mediated isothermal amplification reaction in step 2) is 20 μL: 10 μL of reaction premix, 8 μL of primer set and 2 μL of lysed sample solution;

[0026] When the acid-base indicator in the reaction premix is ​​phenol red, the amplification product will be yellow, indicating that feline herpesvirus was detected in the sample and the sample is positive; if the amplification product is purple-red or pink, it indicates that feline herpesvirus was not detected in the sample or the concentration of feline herpesvirus in the sample did not reach the detection limit.

[0027] The feline herpesvirus detection primer set provided by this invention includes primer pairs FHV-1-F3 / FHV-1-B3, FHV-1-FIP / FHV-1-BIP, and FHV-1-LF / FHV-1-LB. This invention first identifies conserved regions from the feline herpesvirus gene sequence, then excludes regions with complex higher-order structures within the conserved regions, and finally determines the regions suitable for primer design. After primer pair screening, the resulting primer set can accurately and rapidly detect feline herpesvirus, exhibiting high detection repeatability, specificity, and sensitivity.

[0028] This invention provides a non-diagnostic method for detecting feline herpesvirus, which uses the primer set to perform a loop-mediated isothermal amplification reaction. Compared with the detection method using a common PCR detection kit, this method can react at a constant temperature, does not require temperature changes or complex instruments, has a short reaction time, and provides visualized results, making it suitable for rapid on-site detection. Attached Figure Description

[0029] Figure 1 The results of primer screening for feline herpesvirus detection are shown, where 1, 3, and 5 are the first, second, and third primer groups, respectively; 2, 4, and 5 are negative controls.

[0030] Figure 2 This is the sensitivity result for the first primer set of feline herpesvirus detection; the consideration number marked on the tube wall is the final copy number of the positive control in the reaction system.

[0031] Figure 3 The results are from the repeatability test of the first primer set for feline herpesvirus.

[0032] Figure 4 These are the results of specific detection of feline herpesvirus first primer set, where 1-5 are feline parvovirus nucleic acid samples, feline coronavirus nucleic acid samples, feline calicivirus nucleic acid samples, feline calicivirus nucleic acid samples, and feline leukemia nucleic acid samples. Detailed Implementation

[0033] This invention provides a set of primers for feline herpesvirus detection, including primer pairs FHV-1-F3 / FHV-1-B3, FHV-1-FIP / FHV-1-BIP, and FHV-1-LF / FHV-1-LB.

[0034] In this invention, in order to screen primer sets suitable for feline herpesvirus detection, multiple primer pairs were designed using the conserved sequence of feline herpesvirus as a template, as detailed in Table 1.

[0035] Table 1. Primer information for feline herpesvirus detection

[0036]

[0037] In this invention, three primer sets are used to detect feline herpesvirus. Screening results show that the first primer set is most suitable for detecting feline herpesvirus, while the second and third primer sets cannot successfully amplify the virus. Therefore, this invention provides the application of the aforementioned feline herpesvirus detection primer sets in the preparation of feline herpesvirus detection reagents or kits.

[0038] This invention provides a feline herpesvirus detection kit, including the feline herpesvirus detection primer set. The kit preferably also includes a reaction premix. The reaction premix includes 0.67 μL Bst 2.0 WarmStart DNA polymerase (RT-free) and 8.33 μL buffer; the reaction premix also includes an acid-base indicator; the acid-base indicator preferably includes phenol red. In this embodiment, the reaction premix is ​​purchased from NEB Corporation. The kit preferably also includes a sample lysis buffer. The sample lysis buffer is used to lyse the virus in the sample, releasing the nucleic acid. The sample lysis buffer is purchased from Hangzhou Baiyu Biotechnology Co., Ltd.

[0039] This invention provides the application of the feline herpesvirus detection primer set or the kit in the detection of feline herpesvirus for non-diagnostic purposes.

[0040] This invention provides a non-diagnostic method for detecting feline herpesvirus loop-mediated isothermal amplification, comprising the following steps:

[0041] 1) The sample to be tested is pyrolyzed to obtain the pyrolyzed sample solution;

[0042] 2) The lysed sample solution from step 1) is subjected to loop-mediated isothermal amplification under the action of the primer set and the reaction premix in the kit to obtain the amplification product;

[0043] 3) Determine whether the sample to be tested contains feline herpesvirus based on the color of the amplification product described in step 2).

[0044] In this invention, the solution used for lysis is preferably a sample lysis buffer; the volume ratio of the sample to be tested to the sample lysis buffer is preferably 2:5. The lysis condition is 95°C for 5 minutes. The sample to be tested includes feline body fluids, specifically urine, blood, saliva, etc.

[0045] After obtaining the lysed sample solution, the present invention performs a loop-mediated isothermal amplification reaction on the lysed sample solution under the action of the primer set and the reaction premix in the kit to obtain the amplification product.

[0046] In this invention, the preferred reaction system for the loop-mediated isothermal amplification reaction is 20 μL: 10 μL of reaction premix, 8 μL of primer set, and 2 μL of lysed sample solution. The preferred reaction program for the loop-mediated isothermal amplification reaction is 65℃ for 50 min followed by 10℃ for 2 min.

[0047] After obtaining the amplification product, the color of the amplification product is used to determine whether the sample to be tested contains feline herpesvirus.

[0048] In this invention, the principle of determining whether a sample is infected with feline herpesvirus based on the color change of the amplification product is as follows: DNA polymerase generates a large number of protons during the LAMP amplification reaction, causing a decrease in pH. When phenol red indicator is present in the reaction system, the color of the reaction solution changes from pink to yellow. Positive samples show a yellow color after the reaction, while samples without amplification and negative samples show a purple-red / pink color. When the acid-base indicator in the reaction premix is ​​phenol red, a yellow amplification product indicates that feline herpesvirus is detected in the sample, making it a positive sample. A purple-red or pink amplification product indicates that feline herpesvirus is not detected in the sample or that the concentration of feline herpesvirus in the sample does not reach the detection limit.

[0049] The following detailed description, in conjunction with embodiments, illustrates a feline herpesvirus detection primer set, its isothermal detection kit, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] A method for designing and screening primer sets for feline herpesvirus detection

[0052] Feline herpesvirus (FHV) strain sequences were retrieved and downloaded from the GenBank database using the keywords (Feline PanleukopeniaVirus[Title]) AND Completegenome[Title]. Conserved regions of the viral genome were identified through sequence alignment, and regions with complex higher-order structures were excluded. Primer design regions suitable for designing the "Feline Herpesvirus Visual Rapid Isothermal Detection Kit" were selected, and three sets of primers with excellent parameters were designed. Isothermal amplification primers were designed based on the conserved target sequences using appropriate tools. The designed primers were further validated using BLASTn and Primer-BLAST searches. Primer sequences are shown in Table 1.

[0053] Table 1. Primer information for feline herpesvirus detection

[0054]

[0055]

[0056] Primer selection: The optimal primers for detecting feline herpesvirus were selected.

[0057] The three primer sets designed and synthesized were used to prepare a LAMP amplification reaction system with a total volume of 20 μL: 10 μL of reaction premix, 8 μL of primer sets (including 0.5 μL LFHV-1-F3 (10 μM), 0.5 μL LFHV-1-B3 (10 μM), 2 μL LFHV-1-FIP (20 μM), 2 μL LFHV-1-BIP (20 μM), 1 μL LFHV-1-LF (20 μM), 1 μL LFHV-1-LB (20 μM), 1 μL ddH2O), and 2 μL of lysed sample solution. The reaction premix was purchased from NEB and was prepared using Bst2.0 WarmStart DNA polymerase and WarmStart RTx reverse transcriptase in a special low-buffered reaction solution containing a visible pH indicator. Pre-prepared positive and negative samples were used for detection. Reaction procedure: React at 65℃ for 50 min, then at 10℃ for 2 min. Positive samples will turn yellow after reaction, while samples without amplification and negative samples will turn purple-red / pink.

[0058] Table 2. Results of primer screening for feline herpesvirus

[0059] Positive control + - - negative control - - -

[0060] The results above show that, based on the same positive and negative samples, only the first primer set detected positive samples, while the second and third primer sets did not detect positive samples (see...). Figure 1Therefore, the first primer set was chosen for subsequent experiments.

[0061] Example 2

[0062] Detection sensitivity experiment of the first primer set

[0063] A synthetically produced non-infectious feline herpesvirus gene fragment (plasmid) (synthesized by Suzhou Genewise Biotechnology Co., Ltd.) was used as a positive control, with a concentration of 9 × 10⁻⁶. 10 Copy / μL. Mix the positive control sample at a ratio of 1:100 (9 × 10⁻⁶). 8 (copy / μL), 1:1000 (9×10) 7 (copy / μL), 1:10000 (9×10) 6 (copy / μL), 1:100000 (9×10) 5 (copy / μL), 1:1000000 (9×10) 4 (copy / μL), 1:10000000 (9×10) 3 (copy / μL), 1:100000000 (9×10) 2 (copy / μL), 1:1000000000 (9×10) 1 Dilute 8 times (copy / μL). Detect samples at different dilutions using the same batch of kits. Prepare a stock solution as needed; the number of stock solutions (N) = number of reaction samples + 1. Multiply N by 10 μL of the premixed reaction solution and N by 8 μL of the total volume of the first primer set. Mix well and dispense into reaction tubes. Add 2 μL of the lysed sample solution, positive control, or negative control to each reaction tube as labeled. Place the reaction tubes in a constant temperature reactor and incubate at 65℃ for 50 min, then at 15℃ for 2 min. Results are shown below. Figure 2 See Table 3. Table 3 shows that the detection sensitivity for feline herpesvirus is 18 copies / μL.

[0064] Table 3 Summary of feline herpesvirus sensitivity test results

[0065]

[0066]

[0067] Example 3

[0068] Detection repeatability of the first primer set

[0069] Design 3 batches of reagent kits, with 5 kits in each batch. Test 3 nucleic acid positive samples, 3 weakly positive samples, and 3 negative samples respectively according to the following method. Each kit performs 3 inter-batch tests on each sample, and each batch is repeated 3 times.

[0070] See results Figure 3 The positive control showed yellow amplification products in three replicates, the weak positive control showed orange-toned products in three replicates, and the negative control showed pink. The results of the three replicate tests were relatively consistent, indicating that the first primer set had ideal repeatability.

[0071] Example 4

[0072] Specific detection experiment of the first primer set

[0073] Feline parvovirus nucleic acid samples, feline calicivirus nucleic acid samples, feline coronavirus nucleic acid samples, feline herpesvirus nucleic acid samples, feline HIV samples, and feline leukemia nucleic acid samples were used for testing. The samples were lysed using sample lysis buffer at a ratio of 5:2 (volume ratio) and incubated at 95°C for 5 minutes. The preparation of the stock solution and the reaction procedure were the same as in Example 2.

[0074] The results showed that the first primer set yielded negative results for feline calicivirus nucleic acid, feline coronavirus nucleic acid, feline parvovirus nucleic acid, and feline leukemia nucleic acid samples, but only a positive result was obtained for feline herpesvirus nucleic acid samples (see [link]). Figure 4 This indicates that the reagent has good specificity.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A feline herpesvirus detection primer set, characterized by, This includes primer pairs FHV-1-F3 / FHV-1-B3, FHV-1-FIP / FHV-1-BIP, and FHV-1-LF / FHV-1-LB; The nucleotide sequence of the FHV-1-F3 is shown in SEQ ID NO:1; The nucleotide sequence of the FHV-1-B3 is shown in SEQ ID NO:2; The nucleotide sequence of the FHV-1-FIP is shown in SEQ ID NO:3; The nucleotide sequence of the FHV-1-BIP is shown in SEQ ID NO:4; The nucleotide sequence of the FHV-1-LF is shown in SEQ ID NO:5; The nucleotide sequence of the FHV-1-LB is shown in SEQ ID NO:

6.

2. A feline herpesvirus detection kit, characterized in that, Includes the feline herpesvirus detection primer set as described in claim 1.

3. The detection kit according to claim 2, characterized in that, It also includes reaction premix; The reaction premix contains 0.67 μL of Bst2.0 WarmStart DNA polymerase and 8.33 μL of buffer solution; the reaction premix also contains an acid-base indicator.

4. The detection kit according to claim 3, characterized in that, The acid-base indicator includes phenol red.

5. The detection kit according to any one of claims 2 to 4, characterized in that, It also includes sample lysis buffer.

6. The use of the feline herpesvirus detection primer set according to claim 1 in the preparation of feline herpesvirus detection reagents or kits.

7. The use of the feline herpesvirus detection primer set of claim 1 or the kit of any one of claims 2 to 5 in the detection of feline herpesvirus for non-diagnostic purposes.

8. A non-diagnostic method for detecting feline herpesvirus using loop-mediated isothermal amplification, characterized in that, Includes the following steps: 1) The sample to be tested is pyrolyzed to obtain the pyrolyzed sample solution; 2) The lysed sample solution described in step 1) is subjected to loop-mediated isothermal amplification reaction under the action of the primer set described in claim 1 and the reaction premix in the kit described in claim 3 or 4 to obtain the amplification product; 3) Determine whether the sample to be tested contains feline herpesvirus based on the color of the amplification product described in step 2).

9. The method according to claim 8, characterized in that, In step 1), the solution used for lysis is the sample lysis buffer; the volume ratio of the sample to be tested to the sample lysis buffer is 2:

5. The pyrolysis conditions were 95°C for 5 min.

10. The method according to claim 8, characterized in that, Step 2) The reaction system for the loop-mediated isothermal amplification reaction is 20 μL: 10 μL of reaction premix, 8 μL of primer set and 2 μL of lysed sample solution; When the acid-base indicator in the reaction premix is ​​phenol red, the amplification product will be yellow, indicating that feline herpesvirus was detected in the sample and the sample is positive; if the amplification product is purple-red or pink, it indicates that feline herpesvirus was not detected in the sample or the concentration of feline herpesvirus in the sample did not reach the detection limit.

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