A one-tube colorimetric lamp primer composition, kit for detecting cat infectious nasal tracheitis virus and application thereof

Abstract

This invention relates to a one-tube colorimetric LAMP primer composition, kit, and applications for detecting feline infectious rhinotracheitis virus (FLVR), belonging to the field of molecular biology technology. It solves the problems of existing PCR and qPCR-based infectious disease detection methods, which require complex and expensive temperature control equipment, demand highly skilled operators, have long detection times, and are unsuitable for on-site point-of-care testing. The primer composition of this invention includes an outer primer, an inner primer, and a loop primer. The outer primer consists of an upstream primer F3 and a downstream primer B3; the inner primer consists of an upstream primer FIP and a downstream primer BIP; and the loop primer consists of an upstream primer LF and a downstream primer LB, with sequences shown in SEQ ID NO. 1-6, respectively. This primer composition provides rapid detection, high specificity, a detection limit comparable to qPCR, excellent repeatability and stability, requires no specialized equipment, allows for visual result interpretation, and is suitable for on-site point-of-care testing.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a one-tube colorimetric LAMP primer composition, kit, and application for detecting feline infectious rhinotracheitis virus, and particularly to the application of the primer composition in the preparation of a one-tube colorimetric LAMP product for detecting feline infectious rhinotracheitis virus. Background Technology

[0002] Viral infectious diseases are a major threat to the health of pet cats and other felines, and a key focus in veterinary clinics. Currently, the viral infectious diseases with high morbidity, high mortality, and the highest consumption of veterinary resources include feline infectious rhinoconjunctivitis caused by feline calicivirus (FCV), feline infectious rhinotracheitis caused by feline infectious rhinotracheitis virus (FHV-1), and feline panleukopenia virus (FPV). Among them, FHV-1 belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, and Varicellavirus genus, and is an enveloped double-stranded DNA virus. FHV-1 has high species specificity, with pet cats being the main susceptible host, especially young kittens, with a morbidity rate of 100% and a mortality rate as high as 50% in kittens. Infectious feline rhinotracheitis (FHV-1) caused by FHV-1 infection is a common infectious disease of the upper respiratory tract and eyes in felines. It manifests as inflammatory diseases such as upper respiratory tract inflammation, conjunctivitis, and keratitis. Severe infection can lead to death, with a mortality rate of up to 50% in kittens. After recovery, FHV-1 can remain latent in the trigeminal ganglion, optic ganglion, and nasal turbinate, causing latent infection and potentially reactivating to cause persistent infection. The latent infection and lifelong carrier nature of FHV-1 pose significant challenges to its diagnosis and prevention. Currently, FHV-1 is prevalent worldwide. The carrier rate of FHV-1 in domesticated cats is approximately 5.9%–25%, while the carrier rate in cats in animal shelters, pet markets, and stray cats is as high as 29%–52%. In recent years, cases of FHV-1 infection in felines, including cheetahs, Siberian tigers, and lions, have been increasing annually. The high incidence of FHV-1 not only poses a serious threat to the health of pet cats, but also becomes a potential risk factor for other felines, especially wild felines.

[0003] With the development of molecular biology detection technologies, nucleic acid detection technologies, primarily polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR), have been widely applied to the detection of infectious diseases. Among these, qPCR has been designated by the World Organisation for Animal Health as the gold standard for the clinical diagnosis of most pathogens. However, in clinical diagnosis, PCR and qPCR require complex and expensive temperature control equipment, demand highly skilled operators, and have long testing times, making them unsuitable for point-of-care testing (POCT). The development of isothermal nucleic acid detection technology has provided new ideas for the development of POCT diagnostic methods for infectious diseases. Loop-mediated isothermal amplification (LAMP) does not require complex temperature control equipment. Under isothermal conditions of 60-65℃, it utilizes the efficient strand displacement and polymerase activities of Bst DNA polymerase to complete exponential amplification of nucleic acids in a short time. The amplified products can be detected using various detection techniques such as colorimetry, turbidimetry, lateral flow test strips, and fluorescent probes. The LAMP method is simple to operate, highly specific, has sensitivity comparable to qPCR, and provides visible results, and has been widely used for point-of-care detection of pathogenic microorganisms. Therefore, developing a LAMP detection method for FHV-1 is of great significance for the on-site, real-time detection of feline infectious rhinotracheitis. Summary of the Invention

[0004] This invention addresses the technical problems of existing PCR and qPCR-based infectious disease detection methods, such as the need for complex and expensive temperature control equipment, high professional requirements for operators, long detection times, and unsuitability for on-site real-time detection. It provides an application of a one-tube colorimetric LAMP product for detecting feline infectious rhinotracheitis virus.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0006] In a first aspect, the present invention provides a tube-type colorimetric LAMP primer composition for detecting feline infectious rhinotracheitis virus, comprising an outer primer, an inner primer, and a loop primer; The outer primer consists of an upstream primer F3 and a downstream primer B3. The sequence of the upstream primer F3 is shown in SEQ ID NO.1, and the sequence of the downstream primer B3 is shown in SEQ ID NO.2. The inner primer consists of an upstream primer FIP and a downstream primer BIP. The sequence of the upstream primer FIP is shown in SEQ ID NO.3, and the sequence of the downstream primer BIP is shown in SEQ ID NO.4. The loop primer consists of an upstream primer LF and a downstream primer LB. The sequence of the upstream primer LF is shown in SEQ ID NO.5, and the sequence of the downstream primer LB is shown in SEQ ID NO.6.

[0007] Secondly, the present invention provides the use of the above-mentioned primer composition in the preparation of a one-tube colorimetric LAMP product for detecting feline infectious rhinotracheitis virus.

[0008] Preferably, the product is a reagent or a kit.

[0009] Thirdly, the present invention provides a one-tube colorimetric LAMP kit containing the above-described primer composition for detecting feline infectious rhinotracheitis virus.

[0010] Preferably, it also includes LAMP reaction reagents.

[0011] More preferably, the LAMP reaction reagent includes Bst 3.0 polymerase, cresol red, and EvaGreen dye.

[0012] Preferably, it also includes a positive plasmid of the FHV-1 TK gene; The positive plasmid of the FHV-1 TK gene was synthesized by the following method: the complete TK gene sequence was synthesized based on the full genome sequence of the FHV-1C-27 strain registered in GenBank and cloned into the pGEM-T vector.

[0013] Fourthly, the present invention provides a one-tube colorimetric LAMP detection method for feline infectious rhinotracheitis virus (FIRSV) for non-diagnostic purposes using the above-mentioned kit, comprising the following steps: S1, Extract DNA from the sample to be tested; S2, LAMP amplification of the DNA of the sample to be tested using the primer composition; S3, the result is determined by visual colorimetric observation; Alternatively, the results can be determined by visual colorimetric observation and fluorescence amplification curves. The criteria for judging the results of the visual colorimetric observation are as follows: the color of the positive reaction changes from violet to yellow, and the color of the negative reaction remains violet. The criteria for determining the results of the fluorescence amplification curve are as follows: a fluorescence amplification curve exceeding the threshold is considered positive, and a fluorescence amplification curve not exceeding the threshold is considered negative. The threshold is 10 times the standard deviation of the fluorescence value of the negative control in the first 15 cycles.

[0014] Preferably, the LAMP amplification reaction system is 25 μL, comprising 2.5 μL of 10× isothermal amplification buffer, 1.0 μL of 100 mmol / L MgSO4 aqueous solution, 4.0 μL of 10 mmol / L dNTPs aqueous solution, 2.5 μL of 10× LAMP primer mixture, 1 μL of 8000 U / mL Bst 3.0 DNA polymerase aqueous solution, 0.5 μL of 0.65 mmol / L cresol red aqueous solution, 0.5 μL of 50× EvaGreen dye, 2 μL of sample DNA, and nuclease-free water to a final volume of 25 μL. The isothermal diffusion buffer solution comprises 200 mmol / L Tris-HCl, 100 mmol / L (NH4)2SO4, 1.5 mmol / L KCl, 20 mmol / L MgSO4 and 1% Tween-20, pH 8.8. The concentrations of upstream primer F3 and downstream primer B3 are each 2 μmol / L, the concentrations of upstream primer FIP and downstream primer BIP are each 14 μmol / L, and the concentrations of upstream primer LF and downstream primer LB are each 10 μmol / L.

[0015] Preferably, the LAMP amplification reaction temperature is 63°C and the reaction time is 40 min; Preferably, the reaction temperature of the real-time PCR amplification instrument is 63°C, the reaction time is 40 min, and the fluorescence signal is collected once every 30 s through the SYBR Green channel.

[0016] Compared with the prior art, the present invention has the following technical effects: (1) The one-tube colorimetric LAMP primer composition of the present invention for detecting feline infectious rhinotracheitis virus can rapidly detect feline infectious rhinotracheitis virus, and the results can be obtained within 40 minutes. It has high specificity and the lowest detection limit is comparable to that of qPCR.

[0017] (2) The one-tube colorimetric LAMP primer composition of the present invention for detecting feline infectious rhinotracheitis virus adopts closed tube detection, which can avoid aerosol contamination.

[0018] (3) The one-tube colorimetric LAMP kit for detecting feline infectious rhinotracheitis virus of the present invention integrates visual colorimetric detection and real-time fluorescence quantification functions, which can be observed by visual colorimetric observation or detected by fluorescence amplification curve.

[0019] (4) The kit of the present invention provides a one-tube colorimetric LAMP detection method for feline infectious rhinotracheitis virus for non-diagnostic purposes. It is simple and fast to operate, requires no professional equipment, and can realize visual result judgment. It is suitable for the immediate detection of feline infectious rhinotracheitis virus in primary veterinary hospitals and field environments. Attached Figure Description

[0020] Figure 1 This document describes the design and validation of the one-tube colorimetric LAMP primer composition used for detecting FHV-1 in Example 1. (A) shows the fluorescence amplification curves; red, pink, and orange represent the triplet fluorescence amplification curves of the FHV-1 viral DNA template, while blue represents the triplet fluorescence amplification curve of the nuclease-free water template. (B) shows the agarose gel electrophoresis results of the amplification products from two repeated experiments; "+" template represents FHV-1 viral DNA, and "-" template represents nuclease-free water. (C) shows the visual colorimetric observation results; positive reactions are yellow, and negative reactions are violet.

[0021] Figure 2 This is a graph showing the optimized reaction temperature results of the FHV-1 one-tube colorimetric LAMP detection method in Example 2. The top row shows the colorimetric observation results (yellow indicates a positive reaction, and red indicates a negative reaction), and the bottom row shows the fluorescence amplification curves. NTC is the negative control.

[0022] Figure 3 This is a graph showing the optimized primer concentration results for the FHV-1 one-tube colorimetric LAMP detection method in Example 2. The top row shows the colorimetric observation results, and the bottom row shows the fluorescence amplification curves. NTC is the negative control.

[0023] Figure 4 This is a graph showing the optimization results of the loop primer concentration in the FHV-1 one-tube colorimetric LAMP detection method in Example 2. The top row shows the colorimetric observation results, and the bottom row shows the fluorescence amplification curves. NTC is the negative control.

[0024] Figure 5 For the FHV-1 one-tube colorimetric LAMP detection method in Example 2, Mg 2+ The results of concentration optimization are shown in the figure. The top row shows the colorimetric observation results, and the bottom row shows the fluorescence amplification curves. NTC is the negative control.

[0025] Figure 6 This is a graph showing the optimization results of dNTP concentration in the FHV-1 one-tube colorimetric LAMP detection method in Example 2. The top row shows the colorimetric observation results, and the bottom row shows the fluorescence amplification curves. NTC is the negative control.

[0026] Figure 7 This is a graph showing the optimized reaction time of the FHV-1 one-tube colorimetric LAMP detection method in Example 2. Yellow indicates a positive reaction, and red indicates a negative reaction.

[0027] Figure 8 This is a graph showing the specificity analysis results of the FHV-1 one-tube colorimetric LAMP detection method in Example 3. The top row shows the colorimetric observation results (yellow indicates a positive reaction, and red indicates a negative reaction), and the bottom row shows the fluorescence amplification curve. NTC is the negative control.

[0028] Figure 9 This is a graph showing the sensitivity analysis results of the FHV-1 one-tube colorimetric LAMP detection method in Example 4. The top row shows the colorimetric observation results (yellow indicates a positive reaction, and red indicates a negative reaction), and the bottom row shows the fluorescence amplification curve. NTC is the negative control. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. The embodiments given below are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The use of terms as used in this specification is merely for describing particular embodiments and is not intended to limit the scope of this application.

[0031] The positive plasmid of the FHV-1 TK gene used in the example was synthesized by the following method: the complete TK gene sequence was synthesized based on the full genome sequence of FHV-1 C-27 strain registered in GenBank and cloned into the pGEM-T vector. The sequence information can be found in GenBank (GenBank accession number: FJ478159).

[0032] Example 1: Design and validation of LAMP primer set for feline infectious rhinotracheitis virus The TK gene of FHV-1 is highly conserved, exhibiting nearly 100% nucleotide homology among different isolates, and has been widely used as a target gene for developing molecular detection methods for FHV-1. Using the TK gene as the target gene, a primer set for LAMP detection was designed using Primer Explorer Version 5 online software. Primer specificity, dimer formation, and hairpin structure formation were analyzed using Primer Blast and MFE Primer online software. The primer set included two outer primers, two inner primers, and two loop primers. Primer sequences are shown in Table 1.

[0033] Table 1. One-tube colorimetric LAMP primer compositions for detecting FHV-1

[0034] Viral DNA was extracted from cell cultures of the FHV-1 isolate using a viral DNA / RNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The FHV-1 isolate was identified and preserved by the laboratory of Jilin Agricultural Science and Technology College.

[0035] The LAMP amplification reaction system was 25 μL, comprising: 2.5 μL 10× isothermal amplification buffer, 1.0 μL 100 mmol / L MgSO4 aqueous solution, 4.0 μL 10 mmol / L dNTPs aqueous solution, 2.5 μL 10× LAMP primer mixture, 1 μL 8000 U / mL Bst 3.0 DNA polymerase aqueous solution, 0.5 μL 0.65 mmol / L cresol red aqueous solution, and 0.5 μL... 50×EvaGreen dye, 2 μL of DNA sample, and nuclease-free water to bring the volume to 25 μL; the isothermal diffusion buffer consisted of 200 mmol / L Tris-HCl, 100 mmol / L (NH4)2SO4, 1.5 mmol / L KCl, 20 mmol / L MgSO4, and 1% Tween-20, pH 8.8; the concentrations of upstream primer F3 and downstream primer B3 were each 2 μmol / L, the concentrations of upstream primer FIP and downstream primer BIP were each 14 μmol / L, and the concentrations of upstream primer LF and downstream primer LB were each 10 μmol / L.

[0036] Amplification was performed using a LightCycler® 96 instrument, with the reaction timed at 65°C for 45 min. Fluorescence signals were acquired every 30 s via the SYBR Green channel, followed by termination of the reaction by heating at 80°C for 5 min. LAMP amplification was performed using reaction tubes, with closed-tube detection employed.

[0037] LAMP amplification results are determined in the following ways: (1) Fluorescence amplification curve: the threshold is determined based on the fluorescence value of the negative control in the first 15 cycles. Fluorescence amplification curves exceeding the threshold are positive, and fluorescence amplification curves not exceeding the threshold are negative. The intersection of the positive amplification curve and the threshold is the positive time point (Tp); (2) Visual colorimetric observation: the color of the positive reaction changes from violet to yellow, and the negative reaction remains violet; (3) 2% agarose gel electrophoresis analysis: the positive reaction shows characteristic ladder-like bands, and the negative reaction has no bands.

[0038] The extracted FHV-1 DNA was amplified using the designed LAMP primer set, with nuclease-free water as a negative control. The experiment was repeated three times, and the results are as follows: Figure 1 As shown, (A) is the fluorescence amplification curve, (B) is the agarose gel electrophoresis result of the amplification products, and (C) is the visual colorimetric observation result. The fluorescence amplification curve results show that the designed LAMP primers showed an amplification curve in the positive nucleic acid, while no amplification curve was observed in the negative control; the positive nucleic acid electrophoresis detection showed bright ladder-like bands in the amplification products; the colorimetric observation results showed that the positive reaction tube showed a clear color change from violet to yellow, while the negative control remained violet.

[0039] In summary, the primer set designed in this invention can be used for colorimetric LAMP and fluorescent LAMP detection of FHV-1.

[0040] Example 2: Optimization of reaction conditions for the one-tube colorimetric LAMP detection method of FHV-1 1. Optimization of reaction temperature The LAMP amplification reaction system described in Example 1 was used, with FHV-1 DNA extracted in Example 1 as a template. The reaction temperatures were set at 59, 60, 61, 62, 63, 64, and 65°C, and the reaction time was 45 min. The reaction was performed using a LightCycler® 96 instrument. Fluorescence signals were collected every 30 seconds via the SYBR Green channel, followed by heating at 80°C for 5 min to terminate the reaction. Amplification results were determined by fluorescence amplification curves and visual colorimetric observation (other conditions were the same as in Example 1). The real-time fluorescence amplification curves of the LAMP method were visualized as a heatmap using GraphPad Prism 8.2.0 software. Rainbow colors represent fluorescence intensity from low to high, and white indicates fluorescence intensity below the fluorescence threshold. Nuclease-free water was used as a negative control.

[0041] The results are as follows Figure 2 As shown, within the temperature range of 59℃ to 65℃, the positive reaction tubes all exhibited a clear color change from violet to yellow, while the negative control remained violet. The yellow color development was most pronounced at 63℃, 64℃, and 65℃. Fluorescence amplification curves indicated that low Tp values ​​and high endpoint fluorescence values ​​were obtained under these three temperature conditions, with no significant differences. In conclusion, the optimal amplification temperature for the one-tube colorimetric LAMP detection method for feline infectious rhinotracheitis virus was determined to be 63℃.

[0042] 2. Optimization of inner primer concentration According to the LAMP amplification reaction system in Example 1, using FHV-1 DNA extracted in Example 1 as a template, the final concentrations of outer primers F3 and B3 were fixed at 0.2 μmol / L each, and the final concentrations of loop primers LF and LB were fixed at 0.4 μmol / L each. The concentration ratios of inner primer FIP / outer primer F3 were set to 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1, and the concentration ratios of inner primer BIP / outer primer B3 were set to 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and 9:1. The reaction temperature was set to 63℃, and the reaction time was 45 min. The reaction was performed in a LightCycler® 96 instrument. Fluorescence signals were collected every 30 s through the SYBR Green channel, and then the reaction was terminated by heating at 80℃ for 5 min. The amplification results were determined by fluorescence amplification curves and visual colorimetric observation (other conditions were the same as in Example 1). The real-time fluorescence amplification curves of the LAMP method were visualized as a heatmap using GraphPad Prism 8.2.0 software. Rainbow colors represent fluorescence intensity from low to high, while white indicates fluorescence intensity below the fluorescence threshold. Nuclease-free water was used as a negative control.

[0043] The results are as follows Figure 3 As shown, when the concentration ratio is 7:1, the Tp value is the lowest, the endpoint fluorescence intensity is the highest, and the yellow color development in the reaction tube is the most vivid. Therefore, the optimal final concentrations of the inner primers FIP and BIP are determined to be 1.4 μmol / L each.

[0044] 3. Optimization of Loop Primer Concentration Following the LAMP amplification reaction system described in Example 1, using FHV-1 DNA extracted in Example 1 as a template, the final concentrations of outer primers F3 and B3 were fixed at 0.2 μmol / L each, and the final concentrations of inner primers FIP and BIP were fixed at 1.4 μmol / L. The ratios of circular primer LF to outer primer F3 were set to 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1, and the ratios of circular primer LB to outer primer B3 were set to 1:1, 2:1, 3:1, 4:1, 5:1, and 6:1. The reaction temperature was set at 63°C, and the reaction time was 45 min. The reaction was performed using a LightCycler® 96 instrument. Fluorescence signals were collected every 30 seconds through the SYBR Green channel, followed by heating at 80°C for 5 min to terminate the reaction. The amplification results were determined by fluorescence amplification curves and visual colorimetric observation (other conditions were the same as in Example 1). The real-time fluorescence amplification curves of the LAMP method were visualized as a heatmap using GraphPadPrism 8.2.0 software. The rainbow colors represent fluorescence intensity from low to high, while white indicates fluorescence intensity below the fluorescence threshold. Nuclease-free water serves as a negative control.

[0045] The results are as follows Figure 4As shown, when the concentration ratio is 5:1, the Tp value is the lowest, the endpoint fluorescence intensity is the highest, and the yellow color development in the reaction tube is the most vivid. Therefore, the optimal final concentrations of the loop primers LF and LB are determined to be 1.0 μmol / L each.

[0046] 4.Mg 2+ Concentration optimization According to the LAMP amplification reaction system in Example 1, using the FHV-1 DNA extracted in Example 1 as a template, the final concentrations of outer primers F3 and B3 were fixed at 0.2 μmol / L each, the final concentrations of inner primers FIP and BIP were fixed at 1.4 μmol / L each, and the final concentrations of loop primers LF and LB were fixed at 1.0 μmol / L each. Mg was set. 2+ The final concentration was 3–8 mmol / L, with a gradient of 1 mmol / L. The reaction temperature was set at 63°C, and the reaction time was 45 min. The reaction was performed using a LightCycler® 96 instrument. Fluorescence signals were collected every 30 seconds through the SYBR Green channel. The reaction was then terminated by heating at 80°C for 5 min. The amplification results were determined by fluorescence amplification curves and visual colorimetric observation (other conditions were the same as in Example 1). The real-time fluorescence amplification curves of the LAMP method were visualized as a heatmap using GraphPad Prism 8.2.0 software. Rainbow colors represent fluorescence intensity from low to high, and white represents fluorescence intensity below the fluorescence threshold. Nuclease-free water was used as a negative control.

[0047] The results are as follows Figure 5 As shown, Mg 2+ At a final concentration of 6 mmol / L, the Tp value was the lowest, the endpoint fluorescence intensity was the highest, and the yellow color development in the reaction tube was the most vivid. Therefore, the optimal Mg concentration was determined. 2+ The final concentration was 6 mmol / L.

[0048] 5. Optimization of dNTP concentration According to the LAMP amplification reaction system in Example 1, using the FHV-1 DNA extracted in Example 1 as a template, the final concentrations of outer primers F3 and B3 were fixed at 0.2 μmol / L each, the final concentrations of inner primers FIP and BIP were 1.4 μmol / L each, and the final concentrations of circular primers LF and LB were 1.0 μmol / L each. Mg 2+The final concentration was 6 mmol / L. dNTP concentrations were set at 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol / L. The reaction temperature was set at 63°C, and the reaction time was 45 min. The reaction was performed using a LightCycler® 96 instrument. Fluorescence signals were acquired every 30 seconds via the SYBR Green channel, followed by heating at 80°C for 5 min to terminate the reaction. Amplification results were determined by fluorescence amplification curves and visual colorimetric observation (other conditions were the same as in Example 1). The real-time fluorescence amplification curves of the LAMP method were visualized as a heatmap using GraphPad Prism 8.2.0 software. Rainbow colors represent fluorescence intensity from low to high, and white indicates fluorescence intensity below the fluorescence threshold. Nuclease-free water was used as a negative control.

[0049] The results are as follows Figure 6 As shown, the Tp value was lowest and the endpoint fluorescence intensity was highest when the final concentration of dNTPs was 1.6 mmol / L, and the yellow color development in the reaction tube was most vivid. Therefore, the optimal final concentration of dNTPs was determined to be 1.6 mmol / L.

[0050] 6. Optimization of reaction time According to the LAMP amplification reaction system in Example 1, detection was performed using 10,000-fold diluted FHV-1 DNA as a template. The final concentrations of the outer primers F3 and B3 were fixed at 0.2 μmol / L each, the final concentrations of the inner primers FIP and BIP were 1.4 μmol / L each, and the final concentrations of the loop primers LF and LB were 1.0 μmol / L each. Mg 2+ The final concentration was 6 mmol / L, the dNTP concentration was 1.6 mmol / L, the reaction temperature was set at 63℃, and the reaction time was set at 10, 20, 30, 40, 50, and 60 min, respectively. The color change of the reaction tube was observed (other conditions were the same as in Example 1).

[0051] The results are as follows Figure 7 As shown, positive reaction tubes exhibited a color change from red to yellow after 20-60 min of reaction, while no color change was observed after 10 min. The color intensity significantly increased in the 40-60 min range without significant difference, therefore the optimal amplification time was determined to be 40 min.

[0052] In summary, the optimal reaction system for the FHV-1 one-tube colorimetric LAMP detection method was determined, as shown in Table 2. The amplification conditions were 63℃ for 40 min. Amplification could be performed using a metal bath or a water bath. The positive reaction color changed from violet to yellow, while the negative reaction remained violet, observed by the naked eye. Alternatively, a real-time PCR instrument could be used, and the results could be determined by observing the fluorescence amplification curve and visual colorimetric observation.

[0053] Table 2 Optimal reaction system for the FHV-1 one-tube colorimetric LAMP assay

[0054] Example 3: Specificity analysis of the FHV-1 one-tube colorimetric LAMP detection method To evaluate the specificity of the FHV-1 one-tube colorimetric LAMP assay, the optimal reaction system and conditions described in Example 2 were used to detect feline panleukopenia virus (FPV), canine herpesvirus type 1 (CHV-1), pseudorabies virus (PRV), and Bordetella bronchiseptica (Bordeix bacillus). Bb Streptococcus pneumoniae () Sp ), Chlamydia felis ( C.felis ) and feline mycoplasma ( M.felis DNA from clinically positive samples (identified by quantitative real-time PCR), and RNA from clinically positive samples of feline calicivirus (FCV) and feline coronavirus (FCoV). Extracted FHV-1 isolate DNA (FHV-1) and the positive plasmid of the FHV-1 TK gene (pMD-TK) were used as positive controls, and nuclease-free water (NTC) was used as a negative control.

[0055] See results Figure 8 Only the pMD-TK reaction tubes containing the FHV-1 isolate nucleic acid and the FHV-1 TK gene positive plasmid showed yellow fluorescence amplification curves exceeding the threshold. Other tested pathogens remained violet and did not show fluorescence amplification curves exceeding the threshold, consistent with the negative control results. These results indicate that the established LAMP detection method showed no cross-reactivity with any of the tested pathogens, including FCV, FPV, FCoV, CHV-1, PRV, Bordetella bronchiseptica, Streptococcus pneumoniae, Feline Chlamydia, and Feline Mycoplasma, demonstrating its high specificity for FHV-1 detection.

[0056] Example 4: Analysis of the lowest detection limit of the FHV-1 one-tube colorimetric LAMP detection method The positive plasmid of the FHV-1 TK gene was serially diluted 10-fold (1~10) with nuclease-free water. 7 The limit of detection for LAMP assays was determined by detecting a series of diluted plasmids (copies / μL).

[0057] The results are as follows Figure 9 As shown, the LAMP method can detect plasmids down to 100 copies by visual colorimetric observation; while the detection limit can be as low as 10 copies when monitored by real-time fluorescence amplification curve.

[0058] Example 5: Repeatability analysis of the FHV-1 one-tube colorimetric LAMP detection method The positive plasmid of the FHV-1 TK gene was diluted to 10 with nuclease-free water. 2 10 4 and 10 6 The plasmids at three dilution gradients were tested using the optimal reaction system and conditions described in Example 2, with each concentration repeated three times. Three inter-batch assays were also performed. The Tp values ​​and coefficients of variation for both intra-batch and inter-batch assays were statistically analyzed.

[0059] The results are shown in Table 3. Using positive plasmids of the FHV-1 TK gene at different concentrations as templates, intra- and inter-batch experiments were conducted. All plasmid concentrations consistently showed a color change from violet to yellow in repeated tests. The coefficient of variation of Tp values ​​in both intra- and inter-batch experiments was less than 5% at all concentrations, indicating that the established LAMP method has high stability and reproducibility.

[0060] Table 3. Intra-batch and inter-batch testing results

[0061] Example 6: Clinical Sample Testing Eighty-seven clinically suspected samples were simultaneously tested using the established LAMP detection method and the FHV-1 real-time PCR detection kit (Beijing Senkang Biotechnology Development Co., Ltd.). Using the results of the FHV-1 real-time PCR detection kit as a reference standard, the concordance rate, comparative sensitivity, and comparative specificity of the LAMP detection method were calculated using a four-fold table. The Kappa value was analyzed using the chi-square test with SPSS 18.0 software.

[0062] The FHV-1 one-tube colorimetric LAMP detection method established in this invention was used to test 87 nasal swab samples, and 44 samples were positive for FHV-1 nucleic acid. Of the 43 samples that were negative by colorimetric LAMP, 2 were positive by qPCR. Using qPCR as a reference, the specificity of the FHV-1 one-tube colorimetric LAMP detection method established in this invention was 100% (95% confidence interval: 89.9%~100.00%), the sensitivity was 95.7% (95% confidence interval: 82.9%~99.2%), and the overall concordance rate was 97.7% (Table 4). The consistency between the colorimetric LAMP detection method and the qPCR detection results was evaluated using Cohen's kappa statistic, and the kappa value was 0.95, indicating a high degree of consistency between the two methods. In conclusion, the one-tube colorimetric LAMP detection method established in this invention can serve as a reliable point-of-care testing tool for FHV-1 and can be applied to clinical diagnosis.

[0063] Table 4 Clinical Sample Test Results

[0064] In summary, this invention designs a LAMP amplification primer set based on the TK gene of feline infectious rhinotracheitis virus (FIRV) and establishes a one-tube LAMP detection method combining colorimetric and fluorescence methods. This method has the following advantages: (1) closed-tube detection avoids aerosol contamination; (2) it integrates visual colorimetric detection and real-time fluorescence quantification; (3) it is rapid, with results obtained within 40 minutes; (4) it has high specificity; (5) its limit of detection is comparable to qPCR; and (6) it has excellent repeatability and stability. The one-tube colorimetric LAMP detection method for FIRV established in this invention is simple and rapid to operate, requires no professional equipment, and allows for visual result determination. It is suitable for the immediate detection of FIRV in primary veterinary hospitals and field environments.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. All modifications made based on the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A one-tube colorimetric LAMP primer composition for detecting feline infectious rhinotracheitis virus, characterized in that, Including outer primers, inner primers, and loop primers; The outer primer consists of an upstream primer F3 and a downstream primer B3. The sequence of the upstream primer F3 is shown in SEQ ID NO.1, and the sequence of the downstream primer B3 is shown in SEQ ID NO.

2. The inner primer consists of an upstream primer FIP and a downstream primer BIP. The sequence of the upstream primer FIP is shown in SEQ ID NO.3, and the sequence of the downstream primer BIP is shown in SEQ ID NO.

4. The loop primer consists of an upstream primer LF and a downstream primer LB. The sequence of the upstream primer LF is shown in SEQ ID NO.5, and the sequence of the downstream primer LB is shown in SEQ ID NO.

6.

2. The use of the primer composition according to claim 1 in the preparation of a one-tube colorimetric LAMP product for detecting feline infectious rhinotracheitis virus.

3. The application according to claim 2, characterized in that, The product is a reagent or kit.

4. A one-tube colorimetric LAMP kit for detecting feline infectious rhinotracheitis virus containing the primer composition of claim 1.

5. The reagent kit according to claim 4, characterized in that, It also includes LAMP reaction reagents.

6. The reagent kit according to claim 5, characterized in that, The LAMP reaction reagents include Bst 3.0 polymerase, cresol red, and EvaGreen dye.

7. The reagent kit according to claim 4, characterized in that, It also includes positive plasmids for the FHV-1 TK gene; The positive plasmid of the FHV-1 TK gene was synthesized by the following method: the complete TK gene sequence was synthesized based on the full genome sequence of FHV-1 C-27 strain registered in GenBank and cloned into the pGEM-T vector.

8. A one-tube colorimetric LAMP detection method for feline infectious rhinotracheitis virus for non-diagnostic purposes using the kit described in any one of claims 4 to 7, characterized in that, Includes the following steps: S1, Extract DNA from the sample to be tested; S2, LAMP amplification of the DNA of the sample to be tested using the primer composition; S3, the result is determined by visual colorimetric observation; Alternatively, the results can be determined by visual colorimetric observation and fluorescence amplification curves. The criteria for judging the results of the visual colorimetric observation are as follows: the color of the positive reaction changes from violet to yellow, and the color of the negative reaction remains violet. The criteria for determining the results of the fluorescence amplification curve are as follows: a fluorescence amplification curve exceeding the threshold is considered positive, and a fluorescence amplification curve not exceeding the threshold is considered negative. The threshold is 10 times the standard deviation of the fluorescence value of the negative control in the first 15 cycles.

9. The detection method according to claim 8, characterized in that, The LAMP amplification reaction system is 25 μL, including 2.5 μL of 10× isothermal amplification buffer, 1.0 μL of 100 mmol / L MgSO4 aqueous solution, 4.0 μL of 10 mmol / L dNTPs aqueous solution, 2.5 μL of 10× LAMP primer mixture, 1 μL of 8000 U / mL Bst 3.0 DNA polymerase aqueous solution, 0.5 μL of 0.65 mmol / L cresol red aqueous solution, 0.5 μL of 50× EvaGreen dye, 2 μL of sample DNA, and nuclease-free water to bring the volume to 25 μL. The isothermal diffusion buffer solution comprises 200 mmol / L Tris-HCl, 100 mmol / L (NH4)2SO4, 1.5 mmol / L KCl, 20 mmol / L MgSO4 and 1% Tween-20, pH 8.

8. The concentrations of upstream primer F3 and downstream primer B3 are each 2 μmol / L, the concentrations of upstream primer FIP and downstream primer BIP are each 14 μmol / L, and the concentrations of upstream primer LF and downstream primer LB are each 10 μmol / L.

10. The detection method according to claim 8, characterized in that, The LAMP amplification reaction was carried out at a temperature of 63°C for 40 minutes. And / or, the reaction temperature of the real-time PCR amplification instrument is 63℃, the reaction time is 40min, and the fluorescence signal is collected once every 30s through the SYBR Green channel.

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