Cat gamma herpesvirus type 1 isothermal amplification detection primer, kit and application

Abstract

This invention discloses a primer, kit, and application for isothermal amplification detection of feline gamma herpesvirus type 1 (FHV-1), belonging to the field of nucleic acid detection technology. The invention provides primers for detecting FHV-1, comprising one outer primer and one inner primer. The nucleotide sequence of the upstream primer F3 of the outer primer is shown in SEQ ID NO. 2; the nucleotide sequence of the downstream primer B3 of the outer primer is shown in SEQ ID NO. 3; the nucleotide sequence of the upstream primer FIP of the inner primer is shown in SEQ ID NO. 4; and the nucleotide sequence of the downstream primer BIP of the inner primer is shown in SEQ ID NO. 5. The loop-mediated isothermal amplification detection method for FHV established using these primers is rapid, efficient, highly sensitive, and specific. It reduces the diagnostic complexity caused by co-infection, has low equipment dependence, is suitable for field applications, and enables efficient monitoring of viral shedding levels.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, and in particular relates to a primer, kit and application for isothermal amplification detection of feline gamma herpesvirus type 1. Background Technology

[0002] Feline gammaherpesvirus 1 (FcaGHV1) was the first gammaherpesvirus discovered in domestic cats and exhibits the typical latent infection characteristics of gammaherpesviruses. Epidemiological surveys show that the virus has a global distribution. It has been detected in Switzerland and Italy in Europe, Brazil in South America, and Japan and Singapore in Asia, indicating that FcaGHV1 is widespread in cat populations and has become the second most important feline herpesvirus after feline alphaherpesvirus 1 (FHV-1).

[0003] The current detection methods for FcaGHV1 mainly have the following problems: First, it relies on PCR / qPCR technology, which requires sophisticated equipment. All existing reports on FcaGHV1 detection are based on conventional PCR or real-time quantitative PCR. Early studies used degenerate pan-gamma herpesvirus PCR for screening; currently, real-time quantitative PCR targeting the glycoprotein B gene is used for detection. Due to the reliance on sophisticated thermal cyclers and fluorescence detection equipment, the instruments are costly and cumbersome to operate, making them unsuitable for widespread application in field conditions.

[0004] Second, serological tests (such as ELISA) can only reflect past infection history and cannot distinguish between current infection and latent infection, and there is a risk of cross-reactivity; at the same time, the testing cycle is long (several hours), which is not suitable for rapid diagnosis of acute infection.

[0005] Third, existing methods cannot be used to monitor the risk of viral shedding in saliva and environmental transmission. FcaGHV1 is mainly transmitted through saliva, and studies have shown that chronic shedding can occur for at least two months. Monitoring the level of viral shedding in saliva is crucial for developing control measures, but while the qPCR method can be used for quantitative analysis in saliva, it requires sophisticated equipment, is expensive, and is not convenient for frequent implementation in primary care settings such as veterinary clinics, thus hindering real-time tracing of the source of transmission.

[0006] Fourth, no isothermal amplification detection method has yet been established. Loop-mediated isothermal amplification (LAMP) technology has been successfully applied to the detection of various viruses, including FHV-1, which also belongs to the herpesvirus family, for which LAMP detection kits have been reported. However, there is currently no LAMP detection method for FcaGHV1, and LAMP primer sets for both the gB gene and the DNA polymerase gene are lacking. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides isothermal amplification detection primers, kits, and applications for feline gamma herpesvirus type 1 (FGV1) to rapidly, accurately, and efficiently detect FGV1.

[0008] To achieve the above objectives, the present invention provides a primer for isothermal amplification detection of feline gamma herpesvirus type 1, comprising one pair of outer primers and one pair of inner primers; The nucleotide sequence of the upstream primer F3 of the outer primer is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer B3 of the outer primer is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer FIP of the inner primer is shown in SEQ ID NO.4; The nucleotide sequence of the downstream primer BIP of the inner primer is shown in SEQ ID NO.5.

[0009] The present invention also provides a primer for isothermal amplification detection of feline gamma herpesvirus type 1, which may further include a pair of loop primers; The nucleotide sequence of the upstream primer LF of the loop primer is shown in SEQ ID NO.6; The nucleotide sequence of the downstream primer LB of the loop primer is shown in SEQ ID NO.7.

[0010] The present invention also provides a feline gamma herpesvirus type 1 isothermal amplification detection kit, comprising the primers, DNA polymerase, reaction buffer, positive control, negative control and fluorescent detection substance described in the above scheme.

[0011] Preferably, the amount of DNA polymerase in the reaction system is 8-16 U.

[0012] Preferably, the reaction buffer comprises: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 6-8 mM MgSO4, 0.1% Tween 20, 1.4 mM dNTPs, and 0.8-1.0 M betaine.

[0013] This invention also provides the application of the detection kit described above in the detection of feline gamma herpesvirus type 1, comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Amplify the nucleic acid in the sample to be tested using amplification reagents to obtain amplification products; 3) Interpret the results using the fluorescent dye method.

[0014] Preferably, the sample includes one or more of saliva, feces, serum, plasma, blood, tongue swab, throat swab, and nasal swab.

[0015] Preferably, the amplification temperature in step 2) is 60~65℃, and the amplification time is 20~60 minutes.

[0016] Preferably, the fluorescent dye includes Eva Green fluorescent dye or SYBR Green fluorescent dye.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Rapid and efficient. This invention detects FcaGHV1 based on loop-mediated isothermal amplification (LAMP), eliminating the need for the thermal cycling procedure of traditional PCR / qPCR. The entire detection process can be completed within 20 minutes, greatly shortening the reaction time. Furthermore, since the LAMP reaction can complete all amplification under isothermal conditions of 60-65℃, it eliminates the need for the temperature-dependent "denaturation-annealing-extension" cycle, significantly reducing thermal cycling energy consumption and instrument wear, thus improving detection throughput and timeliness.

[0018] (2) High sensitivity and high specificity. LAMP technology relies on four specific primers to recognize six different regions of the target gene, resulting in extremely high detection specificity. For samples with low FcaGHV1 DNA load (such as healthy carrier cats with low viral load in their blood, or asymptomatic cats in the viral incubation period at the time of testing), this invention can effectively avoid false negatives. At the same time, the primer set is designed for six conserved regions of the gB gene, ensuring consistent detection capability for isolates from different geographical locations.

[0019] (3) Low equipment dependence and suitable for field application. LAMP detection is completed under constant temperature conditions of 60~65℃, and rapid detection can be achieved in 20 minutes with only a simple constant temperature heat source and fluorescence detection equipment. The result is interpreted by detecting the reaction status through changes in the collected fluorescence value. There is no need to open the lid, which greatly reduces the possibility of contamination and the occurrence of false positives. This makes the present invention particularly suitable for promotion and use in field environments such as grassroots pet clinics, catteries, breeding farms, stray cat rescue bases, wild feline surveys, and pet transportation quarantine.

[0020] (4) It enables efficient monitoring of viral shedding levels. Compared with qPCR methods that can only detect viral DNA in blood, this invention can be applied to rapid screening of large batches of saliva samples, enabling intensive monitoring of viral shedding levels in individual cats and cat groups. Saliva shedding in cats is the main transmission route of FcaGHV1, and studies have confirmed that persistent chronic shedding can occur for up to two months. This invention can dynamically track these viral shedding peaks in a simple and low-cost manner, which is of outstanding significance in epidemic prevention guidance. It is known that the viral shedding level in feral cats is significantly higher than that in non-feral cats; the LAMP detection method of this invention can assist in evaluating the viral shedding level in feral cats and the effectiveness of culling management measures. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The graph shows the performance of candidate primer set 1. Figure 2 The graph shows the performance of candidate primer set 2. Figure 3 The graph shows the performance of candidate primer set 3. Figure 4 This is a negative test result for candidate primer set 1; Figure 5 This is a sensitivity test plot for candidate primer set 1; Figure 6 This is a specificity test diagram for candidate primer set 1; Figure 7 Image of a negative cat anal swab sample test; Figure 8 Image of a negative swab sample from the mouth, nose, or eyes; Figure 9 Image of a cat with negative anal swabs and oral / nasal / ocular swabs; Figure 10 Image of a negative fecal sample test result from a cat. Figure 11 This is the test image for sample 1; Figure 12 This is the test image for sample 2; Figure 13 This is the test image for sample 3; Figure 14 This is the test image for sample 4; Figure 15 This is the test image for sample 5. Detailed Implementation

[0023] This invention provides a primer for isothermal amplification detection of feline gamma herpesvirus type 1, comprising one pair of outer primers and one pair of inner primers; The nucleotide sequence of the upstream primer F3 of the outer primer is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer B3 of the outer primer is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer FIP of the inner primer is shown in SEQ ID NO.4; The nucleotide sequence of the downstream primer BIP of the inner primer is shown in SEQ ID NO.5.

[0024] In this invention, there are four primers in total, including the inner primer and the outer primer, which respectively identify six different specific regions on the target sequence: designed based on six different sites, namely the F3c, F2c and F1c regions at the 3' end of the target gene and the B1, B2 and B3 regions at the 5' end. The upstream outer primer F3 is complementary to the target gene's F3c region and is used to initiate strand substitution amplification. The downstream outer primer B3 is complementary to the target gene's B3c region and is used to initiate strand substitution amplification in the opposite direction. The upstream inner primer FIP consists of an F2 region and an F1c region, where the F2 region is complementary to the target gene's F2c region, and the F1c region has the same sequence orientation as the target gene's F1c region. It is used to form a circular structure and initiate internal amplification. The downstream inner primer BIP consists of a B2 region and a B1c region, where the B2 region is complementary to the target gene's B2c region, and the B1c region has the same sequence orientation as the target gene's B1c region.

[0025] The present invention also provides a primer for isothermal amplification detection of feline gamma herpesvirus type 1, which may further include a pair of loop primers; The nucleotide sequence of the upstream primer LF of the loop primer is shown in SEQ ID NO.6; The nucleotide sequence of the downstream primer LB of the loop primer is shown in SEQ ID NO.7.

[0026] In this invention, the circular primers LF and LB are complementary to the circular structural regions between the target genes F1c and B1c, respectively, to further improve amplification efficiency and shorten detection time. The addition of circular primers can reduce the detection time of the LAMP reaction from 20 minutes to 10-15 minutes.

[0027] The present invention also provides a feline gamma herpesvirus type 1 isothermal amplification detection kit, comprising the primer set, DNA polymerase, reaction buffer, positive control, negative control and fluorescent detection substance described in the above scheme.

[0028] In this invention, the primer set can be pre-prepared into a 10× or 25× reaction mixture and stored at -20°C for later use.

[0029] The DNA polymerase has strand displacement activity, preferably a large fragment of Bst DNA polymerase or a functionally equivalent variant thereof, and the amount of enzyme in the reaction system is preferably 8-16 U.

[0030] The preferred composition of the reaction buffer is: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 6-8 mM MgSO4, 0.1% Tween 20, 1.4 mM dNTPs, and 0.8-1.0 M betaine; the pH of the Tris-HCl is preferably 8-9, more preferably 8.8.

[0031] The fluorescent detection substance is preferably a fluorescent dye such as Eva Green or SYBR Green, used for result interpretation.

[0032] The positive control is preferably a plasmid DNA containing the target sequence of the FcaGHV1 gB gene, or an extract from an inactivated cat blood / saliva sample that has been confirmed to be FcaGHV1 positive. The positive control plasmid can be constructed as a linearized plasmid DNA containing a conserved region of the FcaGHV1 gB gene.

[0033] The negative control is preferably sterile deionized water or DNA extract from a healthy cat. The healthy cat should be confirmed as FcaGHV1 negative by qPCR testing before it can be used.

[0034] This invention also provides the application of the detection kit described above in the detection of feline gamma herpesvirus type 1, comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Amplify the nucleic acid in the sample to be tested using amplification reagents to obtain amplification products; 3) Interpret the results using the fluorescent dye method.

[0035] Nucleic acid is extracted from the sample to be tested.

[0036] In this invention, the sample includes one or more of the following: saliva, feces, serum, plasma, blood, tongue swab, throat swab, and nasal swab.

[0037] Preferably, the sample extraction method includes the following steps: 1) collecting blood or saliva samples from the cat to be tested; the blood sample is preferably collected using an EDTA anticoagulant tube, approximately 100-200 μL of peripheral blood; the saliva sample is preferably collected by wiping the cat's oral mucosa with a sterile swab and then placing it in a sterile tube containing 500 μL of PBS buffer; 2) extracting total DNA from the sample using a commercially available blood / saliva viral DNA extraction kit or obtaining sample nucleic acid using rapid extraction methods such as thermal lysis / alkali lysis. In this invention, the DNA extraction process must follow the steps in the product instructions to prevent cross-contamination, each extraction step is operated separately, and a negative control sample is added to verify that the extraction process is free from contamination.

[0038] The nucleic acid in the sample to be tested is amplified using amplification reagents to obtain amplification products.

[0039] In the present invention, the amplification method comprises the following steps: using the extracted DNA as a template, adding the LAMP primer set of the present invention to perform loop-mediated isothermal amplification reaction. The amplification reaction system preferably takes a 25 μL system as the optimized scheme, including 1 - 2 μL of template DNA, the LAMP primer mixture preferably has a final concentration of 0.2 μM for each of F3 and B3, a final concentration of 1.6 μM for each of FIP and BIP, 8 - 16 U of Bst DNA polymerase, and the reaction buffer preferably contains 20 mM Tris-HCl with a pH value of 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 6 - 8 mM MgSO4, 0.1% Tween 20, 1.4 mM dNTPs, 0.8 - 1.0 M betaine. If loop primers are added, the final concentration of the loop primers is preferably 0.8 μM, and deionized water is added to make up to 25 μL. The amplification reaction is carried out at a constant temperature of 60 - 65 °C for 20 - 60 minutes.

[0040] The result is judged by using the fluorescent dye method.

[0041] The result is judged by collecting the fluorescence value; When the Tt value of the test sample < 36 and there is a typical S-shaped amplification curve, it is determined that the corresponding item is positive; When the Tt value of the test sample is within the range of 36 - 40, the test result is carried; When the test sample has no Tt value, or the Tt value is within the reference range but there is no typical S-shaped amplification curve, the test result is negative.

[0042] Result description: The Tt value is the time to detect the target gene. The larger the Tt value, the weaker the concentration of the target gene. When the detected Tt value < 16, the concentration is strong; when 16 < Tt value < 26, the concentration is medium; when 26 < Tt value < 36, the concentration is weak.

[0043] Example 1 The present invention selects the glycoprotein B (gB) gene in the FcaGHV1 genome as the detection target. The gB gene is highly conserved in gamma herpesviruses, with a homology of more than 99.5% in different geographical isolates of FcaGHV1 (such as the different isolates found in the United States, Switzerland, Italy, Brazil, Canada, etc. as described above), and the FcaGHV1 gB sequence has extremely low sequence diversity and > 99.5% nucleotide identity globally, making it suitable as a universal detection target.

[0044] By designing a specific LAMP primer set, a rapid, sensitive and specific isothermal detection method for FcaGHV1 is established to achieve rapid screening of FcaGHV1 DNA in blood and saliva samples.

[0045] The nucleotide sequence of FcaGHV gB is shown in SEQ ID NO.1.

[0046] Three sets of primers (including outer primers F3 and B3; inner primers FIP and BIP; and loop primers LF and LB) were designed for the target sequence using the online software Primer Explorer v5, and these three sets of primers were used for LAMP preliminary experiments.

[0047] By comparing the sensitivity and specificity of each primer group, an optimal primer group was finally selected. The primer sequences are shown in Tables 1-3.

[0048] Table 1 Candidate Primer Set 1

[0049] Table 2 Candidate Primer Set 2

[0050] Table 3 Candidate Primer Set 3

[0051] An equal volume of FcaGHV plasmid with a template concentration of 100 copies / μL was used for LAMP reaction. The reaction program was: 63℃, 15s / 63℃, 15s (fluorescence sampling), for a total of 40 cycles, with a total reaction time of 20 minutes. Fluorescence values ​​were detected using a Kiddite T-6 rapid gene analyzer to test the performance of three candidate primer sets for FcaGHV.

[0052] The results show that ( Figures 1-3 Primer set 1 has better detection performance, and gene fragment sequences amplified using primers in primer set 1 were compared with gene sequences and found to have 100% homology with the glycoprotein B gene in the FcaGHV1 genome.

[0053] Example 2 Negative Test The LAMP reaction was performed under the following conditions, with each 25 μL reaction system containing: 2.5 μL 10× Buffer, 0.1–0.4 μM primers F3 and B3, 0.5–4 μM primers FIP and BIP, 0.3–3 μM primers LF and LB, and 0.2–2 μL Eva-Green; Preferred primers are 0.15~0.25μM primers F3 and B3, 1~2μM primers FIP and BIP, 0.5~2μM primers LF and LB, and 0.3~1μL Eva-Green; Using water as a template, a LAMP reaction was performed. Fluorescence values ​​were detected using the Kiddite T-6 rapid gene sequencing instrument. A negative reaction was tested for primer set 1. The results are as follows: Figure 4 As shown.

[0054] The results showed that primer set 1 yielded good negative results.

[0055] Example 3 Sensitivity Test Perform 10-fold serial dilutions on the FcaGHV plasmid DNA, and take 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 Samples at four concentration gradients (copy / μL), 1×10⁻⁶ 2 copies / μL, 1×10 1 Two parallel tests were performed at concentrations of copies / μL. A LAMP reaction was conducted in PCR tubes, and fluorescence values ​​were detected using a Kiddite T-6 rapid gene analyzer to test the detection sensitivity. Results are as follows: Figure 5 As shown.

[0056] The results showed that the primer set had a detection limit of 10 copies of the target nucleic acid within 20 minutes.

[0057] Example 4 Specificity Test Take an equal volume of template concentration 1×10 5 LAMP reactions were performed using copies / μL of feline alpha herpesvirus, feline calicivirus, feline mycoplasma, feline chlamydia, and feline boretula nucleic acid. Fluorescence values ​​were detected using a Kiddit T-6 rapid gene detection system to test whether the primers for feline gamma herpesvirus would exhibit nonspecific reactions with other feline respiratory pathogens. Results are as follows: Figure 6 As shown.

[0058] The results showed that the feline gamma herpesvirus primers designed in this invention exhibited positive amplification curves for feline gamma herpesvirus samples and negative amplification curves for samples containing feline alpha herpesvirus, feline calicivirus, feline mycoplasma, feline chlamydia, and feline borénezolophilus. This indicates that the feline gamma herpesvirus detection method established in this invention does not exhibit nonspecific reactions with feline alpha herpesvirus, feline calicivirus, feline mycoplasma, feline chlamydia, and feline borénezolophilus, demonstrating that the primers provided in this invention have good specificity.

[0059] Example 5 Anti-interference test Negative samples were collected from different parts of the cat for LAMP reaction to detect whether the detection results of primer set 1 were affected by the samples. The results are as follows: Figures 7-10 As shown.

[0060] The results showed that the test was negative, indicating that primer set 1 was not affected by the sample.

[0061] Example 6: Clinical Sample Testing Feline gamma herpesvirus-positive samples with respiratory diseases were collected for testing. The reaction system was prepared according to Example 2, and the accuracy of the detection results of primer set 1 was tested. The results are as follows: Figures 11-15 As shown.

[0062] Table 4 Clinical Sample Test Results

[0063] The results are shown in Table 4. Primer set 1 had an accuracy of 100% and a specificity of 100%.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention 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. A primer for isothermal amplification detection of feline gamma herpesvirus type 1, characterized in that, Includes one pair of outer primers and one pair of inner primers; The nucleotide sequence of the upstream primer F3 of the outer primer is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer B3 of the outer primer is shown in SEQ ID NO.3; The nucleotide sequence of the upstream primer FIP of the inner primer is shown in SEQ ID NO.4; The nucleotide sequence of the downstream primer BIP of the inner primer is shown in SEQ ID NO.

5.

2. The primer according to claim 1, characterized in that, It may also include a pair of circular primers; The nucleotide sequence of the upstream primer LF of the loop primer is shown in SEQ ID NO.6; The nucleotide sequence of the downstream primer LB of the loop primer is shown in SEQ ID NO.

7.

3. A feline gamma herpesvirus type 1 isothermal amplification detection kit, characterized in that, It includes the primer set, DNA polymerase, reaction buffer, positive control, negative control and fluorescent detection substance as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The amount of DNA polymerase in the reaction system is 8-16 U.

5. The reagent kit according to claim 3, characterized in that, The reaction buffer solution comprises: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 6-8 mM MgSO4, 0.1% Tween 20, 1.4 mM dNTPs, and 0.8-1.0 M betaine.

6. The use of the detection kit according to any one of claims 3 to 5 in detecting feline gamma herpesvirus type 1, characterized in that, Includes the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Amplify the nucleic acid in the sample to be tested using amplification reagents to obtain the amplification product; 3) Interpret the results using the fluorescent dye method.

7. The application according to claim 6, characterized in that, The samples include one or more of the following: saliva, feces, serum, plasma, blood, tongue swabs, throat swabs, and nasal swabs.

8. The application according to claim 6, characterized in that, The amplification temperature in step 2) is 60~65℃, and the amplification time is 20~60 minutes.

9. The application according to claim 6, characterized in that, The fluorescent dyes include Eva Green fluorescent dye or SYBR Green fluorescent dye.

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