Kit and method for rapidly detecting and diagnosing feline calicivirus

Abstract

The invention discloses a kit and a method for rapidly detecting and diagnosing feline calicivirus. The kit comprises composite magnetic beads, a reverse transcription system and a fluorescent quantitative PCR (Polymerase Chain Reaction) detection system, the composite magnetic bead is Fe3O4-coated UiO-66, the reverse transcription system comprises a reverse transcription primer and a reverse transcription reagent, and the fluorescent quantitative PCR detection system comprises an amplification primer and a reaction reagent. Composite magnetic beads are synthesized from an MOFs material and magnetic nanoparticles through a solvothermal method, then reverse transcription primers are pre-anchored on the surfaces of the composite magnetic beads, and a one-step mRNA detection platform is derived; after the feline calicivirus is hybridized with the surface anchor primer, reverse transcription is immediately carried out, the generated cDNA can be directly amplified through quantitative polymerase chain reaction, and quantitative analysis can be realized without pre-RNA extraction. Compared with the traditional kit, the method has the advantages of simple sample pretreatment, less time consumption, low cost, no need of using toxic organic reagents such as chloroform in the traditional nucleic acid extraction method, no need of tedious steps such as centrifugation and elution, safety and environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a rapid detection and diagnosis kit and method for feline calicivirus. Background Technology

[0002] Feline calicivirus (FCV) is an important pathogen infecting felines. It is a single-stranded RNA virus that can cause severe oral ulcers, respiratory diseases, and fatal systemic infections, posing a persistent threat to pet health. The virus is highly contagious and has a large number of asymptomatic carriers, making its spread difficult to prevent and control. Therefore, developing rapid and accurate detection technologies is crucial for FCV prevention and control.

[0003] Currently, FCV detection methods mainly include virus isolation, serological testing, and molecular biological detection. Virus isolation is the gold standard for diagnosing FCV infection, but this method is complex, time-consuming, and requires high-level experimental conditions and techniques. While serological testing is relatively simple, its sensitivity and specificity are limited, and it cannot differentiate between infection and vaccine immunization. Molecular biological detection methods, such as reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), have advantages such as high sensitivity and specificity, and have become the main means of FCV detection. However, these methods are still limited by complex operation or cumbersome sample pretreatment steps, restricting their widespread application. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an FCV extraction and detection method that is simple, fast, low-cost, sensitive and does not require a separate nucleic acid extraction step, thereby facilitating the early and rapid detection of FCV virus.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A kit for detecting and diagnosing feline calicivirus includes composite magnetic beads, a reverse transcription system, and a real-time PCR detection system. The composite magnetic beads are Fe3O4@UiO-66; The reverse transcription system includes reverse transcription primers and reverse transcription reagents, and the nucleotide sequence of the reverse transcription primers is: 5'-ATGGCATGGTTGGGGCAGAC-3'.

[0006] Furthermore, the reverse transcription reagent includes an RNase inhibitor, M-MLV reverse transcriptase, and RT buffer.

[0007] Furthermore, the fluorescence quantitative PCR detection system includes amplification primers and reaction reagents.

[0008] Furthermore, the amplification primers include an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is 5'-CGTGATCCGAGCAGGAGGTG-3', and the nucleotide sequence of the downstream primer is 5'-ATGGCATGGTTGGGGCAGAC-3'.

[0009] Furthermore, the reaction reagents include SYBR qPCR Mix.

[0010] In this invention, the composite magnetic beads are core-shell structured composite magnetic beads Fe3O4@UiO-66 formed by metal-organic frameworks (MOFs) and magnetic nanoparticles (Fe3O4). They are prepared using the method described in patent CN118291449A.

[0011] A method for detecting and diagnosing feline calicivirus, a method for non-disease diagnostic purposes, comprising the following steps: Step 1: Mix the composite magnetic bead suspension and the reverse transcription primer buffer to obtain a complex of reverse transcription primers and composite magnetic beads; Step 2: Mix the complex from Step 1 with the sample lysis buffer containing viral RNA and incubate. Step 3: Add reverse transcription reagent to the mixture from Step 2 to reverse transcribe viral RNA into cDNA on the surface of the composite magnetic beads. Step 4: Separate the composite magnetic beads in the reaction solution of Step 3 by applying an external magnetic field, take the supernatant for fluorescence quantitative PCR detection, and obtain the detection results.

[0012] Further, in step 1, the concentration of the composite magnetic beads in the composite magnetic bead suspension is 2 mg / mL, the concentration of the reverse transcription primers in the reverse transcription primer buffer is 10 μM, and the volume ratio of the composite magnetic bead suspension to the reverse transcription primer buffer is 2:1.

[0013] Furthermore, in step 2, the incubation conditions are 4°C for 10 min.

[0014] Furthermore, in step 3, the reverse transcription conditions are 50℃ for 15 min and 85℃ for 2 min.

[0015] Further, in step 4, the system for quantitative real-time PCR detection is as follows: 5 μL of SYBR qPCR Mix, 0.2 μL of 10 μM FCV-F, 0.2 μL of 10 μM FCV-R, 2 μL of sds-cDNA, and ddH2O added to 10 μL; the amplification conditions are 95℃ for 30 s, 95℃ for 10 s, and 60℃ for 30 s, for a total of 45 cycles.

[0016] like Figure 1 As shown, the detection principle of the kit of the present invention is as follows: taking advantage of the structural diversity, high specific surface area, and high adsorption affinity of MOFs, a multifunctional composite magnetic bead is synthesized with magnetic nanoparticles via a solvothermal method. Subsequently, reverse transcription (RT) primers are pre-anchored on the surface of the composite magnetic beads, resulting in a one-step mRNA detection platform. Feline calicivirus hybridizes with the surface-anchored primers and immediately undergoes reverse transcription. The resulting cDNA can be directly amplified by quantitative polymerase chain reaction (qPCR), achieving quantitative analysis without prior RNA extraction.

[0017] The core principle of the mRNA extraction-free detection method used in this invention lies in utilizing the electric double layer (EDL) formed on the surface of metal-organic framework (MOF) materials, such as... Figure 2 As shown, the key role of EDL at the MOF-solution interface in regulating nucleic acid adsorption, recognition, and desorption is elucidated. Primers assembled on the MOF surface can selectively hybridize with target mRNA in the EDL sliding plane. The hybridization of primers and mRNA can be regulated by the thickness of the EDL layer. After primer recognition of the target mRNA, qPCR can be performed without total RNA extraction, enhancing the potential for rapid on-site mRNA detection and making it an ideal choice for point-of-care testing (POCT) applications, thereby achieving efficient capture, recognition, and release of target mRNA.

[0018] The specific mechanism is as follows: 1. Formation and regulation of EDL: EDL is a key factor mediating MOF-nucleic acid interactions. Three nanoscale MOFs (MIL-101, UiO-66, and Zn2Ph2Da) were selected. MIL-101 and UiO-66 contain incompletely coordinated Cr³⁺ and Zr³⁺ on their surfaces, respectively, enabling them to form strong EDL layers and exhibit robust coordination with the phosphate groups of nucleic acids. In aqueous solution, the ionized diffusion layer (EDL) generated by positively charged MOFs is negligible; in buffer solutions (such as PBS), positively charged MOFs (such as UiO-66) electrostatically interact with anions (such as HPO4²⁻ / H2PO4⁻) in the buffer solution, forming an EDL structure consisting of a positively charged Stern layer and a diffused ion layer, as shown below. Figure 3 As shown; The thickness and strength of EDL can be controlled by the ionic strength of the buffer: at low ionic strength (0.01 mM PBS), the EDL is thinner and cannot induce nucleic acid release; at high ionic strength (10 mM PBS), the EDL is thicker and promotes nucleic acid desorption.

[0019] 2. mRNA recognition and hybridization: UiO-66 exhibits a strong charge density layer effect, making it more suitable for detecting mRNA by assembling reverse transcription primers on its surface. This modification allows the target mRNA to be directly captured in the charge density layer region, where reverse transcription occurs on the sliding plane, followed by qPCR amplification.

[0020] Specific reverse transcription primers (RT primers) are adsorbed onto the surface of MOFs to form MOF-primer complexes; Within the slipping plane of EDL, single-stranded primers hybridize with the target mRNA to form a double-stranded structure, such as... Figure 4 As shown; After the double strands are formed, the hydrogen bonds and π-π stacking interactions between the nucleic acid bases and the MOF surface are blocked, leading to their desorption on the MOF surface and enabling selective recognition and release.

[0021] 3. Extraction-free direct detection: By utilizing the EDL effect on the surface of MOFs, target mRNA can be captured directly from lysate, serum, or cell samples without the need for traditional RNA extraction steps.

[0022] By combining magnetic MOF composite materials (such as Fe3O4@UiO-66), magnetic separation and enrichment can be achieved, further improving detection efficiency and ease of operation.

[0023] Because primer amplification is performed on the surface of MOFs, this workflow is called surface primer quantitative polymerase chain reaction (SP-qPCR), establishing a platform for efficient and rapid detection of feline calicivirus. Rapid magnetic separation is achieved through an external magnetic field, and the obtained supernatant is then amplified and analyzed. This method avoids sample pretreatment with toxic organic reagents such as chloroform and cumbersome procedures like centrifugation and elution, thus offering advantages such as low cost, environmental safety, and rapid efficiency. This SP-qPCR method has high practical application value for the detection of feline calicivirus, improving detection sensitivity.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. Fe3O4@UiO-66 composite magnetic beads are used as viral mRNA capture and reverse transcription vectors. They have high specific surface area, strong adsorption capacity and superparamagnetism, and can be quickly used for magnetic separation by applying an external magnetic field. 2. By using surface-anchored reverse transcription primers, the "extraction-reverse transcription-detection" process is integrated, eliminating the cumbersome steps such as centrifugation and elution in traditional RNA extraction, thus improving detection efficiency and greatly reducing costs; 3. Establish a “surface primer quantitative polymerase chain reaction (SP-qPCR)” platform. The sample is lysed by sonication only. The reverse transcription primers are electrostatically adsorbed on the surface of the magnetic composite material. The target mRNA can be directly extracted and enriched from the sample lysate and reverse transcribed in situ. The cDNA is automatically released from the surface of the magnetic beads during the reverse transcription process, without affecting the subsequent amplification.

[0025] This invention utilizes metal-organic frameworks (MOFs) as carriers, leveraging the multiple adsorption properties of aptamers on MOFs. RT-qPCR is performed using MOFs functionalized with aptamer primers that selectively hybridize with viral RNA in biological samples. Nucleic acid amplification is initiated directly on the MOF surface, allowing for quantitative analysis without prior RNA extraction. This method is known as surface primer quantitative polymerase chain reaction (SP-qPCR). Compared to traditional kits (magnetic bead method, viral RNA / DNA extraction kits), sample pretreatment is simpler, less time-consuming, and less costly. It eliminates the need for toxic organic reagents such as chloroform used in traditional nucleic acid extraction methods, as well as cumbersome steps like centrifugation and elution, making it safe and environmentally friendly. This invention can detect viruses at concentrations as low as 1.91 copies / μL, exhibiting high sensitivity. The detection accuracy in actual cat serum samples reaches 93%, making it of significant practical importance for the detection of feline calicivirus. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the principle of the kit for rapid detection of feline calicivirus according to the present invention.

[0027] Figure 2 This is a schematic diagram illustrating the principle of MOFs recognizing mRNA.

[0028] Figure 3 The particle size distribution and zeta potential curves of MIL-101, UiO-66 and Zn2Ph2Da in H2O and PBS are shown.

[0029] Figure 4 This is a schematic diagram of nucleic acid adsorption and desorption on the surface of MOFs.

[0030] Figure 5 This is a TEM image of Fe3O4@UiO-66.

[0031] Figure 6 The hysteresis loop diagram of Fe3O4@UiO-66.

[0032] Figure 7 The image shows the screening results for the optimal concentration of Fe3O4@UiO-66.

[0033] Figure 8 The figure shows the results of the optimal reverse transcription primer selection for the SP-qPCR method.

[0034] Figure 9 The results show the optimization of primer concentration, annealing temperature, and extension time conditions for the SP-qPCR method.

[0035] Figure 10 The results are from the sensitivity test of the kit of the present invention.

[0036] Figure 11 This is a comparison of the effectiveness of the kit method of the present invention with other different methods for the detection of feline calicivirus. Detailed Implementation

[0037] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0040] 1. Preparation of composite magnetic beads Fe3O4@UiO-66 FeCl3·6H2O (0.301 g) and citric acid dihydrate Na3C6H5O7·2H2O (0.18 g) were dissolved in 13.5 mL of ethylene glycol and stirred until completely dissolved. Sodium acetate CH3COONa (0.87 g) was then added and stirred vigorously for 0.5 h. The resulting solution was then transferred to a polytetrafluoroethylene-lined stainless steel high-pressure reactor, sealed, and reacted at 200 °C for 8 h. After cooling to room temperature, the product was collected by an external magnetic field and washed three times each with ethanol and ultrapure water. Finally, the product was vacuum dried at 60 °C for 12 h to obtain Fe3O4 nanoparticles (Fe3O4NPs) for later use.

[0041] Fe3O4@UiO-66 composite material was prepared by a solvothermal method. First, ZrCl4 (0.156 g), H2BDC (0.132 g), and Fe3O4NPs (0.2 g) were weighed and dispersed in 50 mL of N,N-dimethylformamide. Then, 4 mL of HAc was added to the suspension. After mechanical stirring for a few minutes, the suspension was transferred to a polyvinyl fluoride-lined stainless steel autoclave and heated at 130 °C for 12 h. Subsequently, it was washed three times with ethanol and deionized water, and then collected using a magnet. Finally, it was vacuum dried at 60 °C for 12 h to obtain Fe3O4@UiO-66 for later use.

[0042] 2. Construction of a rapid detection and diagnosis kit for feline calicivirus To demonstrate that rapid, in-situ nucleic acid detection can be achieved on the MOF surface, an SP-qPCR method was constructed by pre-anchoring reverse transcription primers to Fe3O4@UiO-66 (e.g., Figure 1 (As shown). The process is as follows: A composite magnetic bead is mixed with reverse transcription primers. The reverse transcription primers electrostatically adsorb onto the surface of the composite magnetic bead, forming a capture probe. This probe is then added to a nucleic acid sample containing a positive sample. The target mRNA is captured by the reverse transcription primers, initiating a reverse transcription reaction in situ on the surface of the composite magnetic bead. The generated double-stranded cDNA detaches from the surface of the composite magnetic bead. Rapid magnetic separation is achieved by applying an external magnetic field. The supernatant is then amplified and analyzed to detect the target mRNA.

[0043] First, composite magnetic beads were prepared using a solvothermal method. TEM experimental results (e.g.) were obtained. Figure 5 The results show that it has a uniform core-shell structure, proving the successful synthesis of Fe3O4@UiO-66. The hysteresis curve results of this material (e.g.) Figure 6 As shown in the figure, this demonstrates that the magnetic bead composite has good paramagnetism and can be used for rapid magnetic separation.

[0044] Next, the adsorption efficiency and concentration of Fe3O4@UiO-66 for reverse transcription primers were optimized and verified. The adsorption efficiency of Fe3O4@UiO-66 for reverse transcription primers was verified by 2% agarose gel electrophoresis. Specifically, 5 μL of 10 μM reverse transcription primers were mixed with different volumes of 5 mg / mL Fe3O4@UiO-66 suspensions, and diluted with DEPC water to a final volume of 20 μL, resulting in solutions with Fe3O4@UiO-66 concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg / mL. The mixtures were incubated at room temperature for 5 minutes, and then adsorbed and precipitated using an external magnetic field. 5 μL of the supernatant was collected for later use.

[0045] Place 0.4 g agarose in an Erlenmeyer flask, add 20 mL of 0.5×TAE electrophoresis buffer, and boil on a hot plate until the agarose is completely dissolved. Cool to approximately 60°C, add 2 μL of nucleic acid dye, mix well, insert the comb, pour the gel into a gel casting apparatus, and after the gel solidifies, remove the comb and transfer it to an electrophoresis tank. Add 1000 mL of 0.5×TAE electrophoresis buffer. Take 5 μL of the supernatant as a sample solution, add 1.0 μL of 6×loading buffer, mix well, and transfer to the sample well. Simultaneously, add 3.0 μL of DNA marker to another sample well as a control. Finally, turn on the power supply at a constant voltage of 120 V and a current of 400 mA for approximately 40 minutes (stop the power supply when the indicator front approaches the edge of the gel). Remove the gel and observe the results using a Tanon gel imaging system. Figure 7 As shown in the figure, channels 1-7 are the supernatants of Fe3O4@UiO-66 mixed with reverse transcription primer solutions at different concentrations ranging from 0 mg / mL to 3 mg / mL. As can be seen from the figure, when the concentration of Fe3O4@UiO-66 reaches 2.0 mg / mL, the band in channel 5 disappears, proving that this concentration of Fe3O4@UiO-66 has adsorbed all the primers, indicating that the adsorption effect of the magnetic MOF is very good.

[0046] Three 10 μM candidate primers—upstream primer (FCV-F, 5 μL), downstream primer (FCV-R, 5 μL), and Oligo dT (15 bp, 5 μL)—were tested in SP-qPCR experiments. Figure 8 As shown, the optimal reverse transcription primer was FCV-R.

[0047] 3. Kit operation and results Fe3O4@UiO-66 (2 mg / mL) was mixed with reverse transcription primer buffer (10 μM, including 10 mM Tris-TE buffer, 0.1 mM EDTA, pH 8.0) at a volume ratio of 2:1 and incubated at room temperature for 5 min. The reverse transcription primers electrostatically adsorbed Fe3O4@UiO-66 onto the surface, forming a virus capture probe. This probe was then co-incubated with a nucleic acid sample containing a positive sample at 4 °C for 10 min. After the target FCV mRNA was captured by the reverse transcription primers, reverse transcription reagent (1 μL RNase inhibitor, 1 μL M-MLV reverse transcriptase, 2 μL RT buffer and 16 μL DEPCwater) was added to initiate a reverse transcription reaction in situ on the MOF surface (reverse transcription program: 50 °C for 15 min, 85 °C for 2 min). The generated double-stranded cDNA detached from the magnetic bead surface. Rapid magnetic separation was achieved by applying an external magnetic field. A SYBR Green qPCR detection system was established (5 μL of SYBR qPCR Mix, 0.2 μL FCV-F (10 μM), 0.2 μL FCV-R (10 μM), 2 μL of sds-cDNA, and then ddH2O was added to 10 μL). The supernatant was amplified and analyzed. The amplification reaction program was: 95 °C for 30 s; 45 cycles of 95 °C for 10 s and 60 °C for 30 s.

[0048] 4. Optimization of SP-qPCR detection conditions To improve the sensitivity of feline calicivirus detection, based on the above experiments, primer concentration, annealing temperature, and extension time were optimized by controlling a single variable. For example... Figure 9 As shown, the optimal primer concentration is 200 nM, the optimal annealing temperature for feline calicivirus detection is 60 °C, and the optimal extension time is 30 s.

[0049] 5. Reagent kit sensitivity test The viral template lysis solution at a concentration of 6.1 × 10⁻⁶ copies / μL was serially diluted two-fold with enzyme-free water, resulting in a viral concentration range of 6.1 × 10⁻⁶ to 1.91 copies / μL. To ensure the accuracy of the test results, the dilution series were used as templates, and a negative control was also included. SP-qPCR was performed three times independently using this method, and the data were analyzed using GraphPadPrism 8.0.1 software.

[0050] Experimental results are as follows Figure 10As shown, there is a good linear relationship between the viral sample concentration and the Ct value. The standard curve is y = -1.908lgC + 39.77. The detection limit of this method is as low as 1.91 copies / μL.

[0051] 6. Reproducibility test of the reagent kit The intra- and inter-assay reproducibility of this method for high, medium, and low concentrations of FCV RNA (30.5–7.63 copies / μL) was evaluated by independent runs on different dates, in triplicate. The coefficient of variation (CV) was calculated based on the obtained Ct values.

[0052]

[0053] The experimental results are shown in the table above. The coefficients of variation (CV) of the kit within and between groups are both less than 5%, indicating good stability.

[0054] 7. Reagent kit addition and recovery experiment To further verify the accuracy and practicality of the developed method, serum samples from healthy cats (purchased from Shanxi Junma Biotechnology Co., Ltd.) were pretreated (4℃, 10000 g, 10 min). 50 µL of the supernatant was diluted 1:15 with Tris-HCl (pH 7.4) and heated (95℃, 10 min) to inactivate DNases. Subsequently, different amounts of FCV targets were added to aliquots for subsequent analysis.

[0055]

[0056] The test results are shown in the table above. The recovery rate of the kit is 82%-101%, which is within the allowable range and meets the requirements. Example 2

[0057] Comparison experiment between the reagent kit and commercial reagent kit Using FCV inactivated virus positive quality control samples as samples, total RNA was extracted and three methods were used: (1) SteadyPure viral DNA / RNA commercial extraction kit, (2) sonication lysis for 10 min (the sample was directly sonicated for 10 min and then subjected to routine PCR detection), and (3) SP-qPCR method to pre-treat and detect FCV virus samples to verify the feasibility of the method.

[0058] FCV viral nucleic acid liquid control sample (purchased from Wuhan Gray Algae Biotechnology Co., Ltd.) frozen at -80°C with a concentration of 6.1 × 10 copies / μL was thawed, and RNA was extracted using the SteadyPure Viral DNA / RNA Extraction Kit (Aike, Hunan, China) and sonication for 10 min. The extracted RNA was stored at -20°C.

[0059] Following the manufacturer's instructions, feline calicivirus RNA was extracted using the SteadyPure Viral DNA / RNA Extraction Kit. The extracted RNA was washed with nuclease-free water and stored at -20°C for later use.

[0060] The results are as follows Figure 11 As shown, the method of this invention detected higher FCV expression levels, and this SP-qPCR method eliminates RNA extraction, allowing for the selective identification and enrichment of mRNA directly from biological samples (such as blood, cells, or tissue lysates) using Fe3O4@UiO-66. This omits the additional mRNA extraction step (1 h), integrating the "capture-enrichment-amplification" process into a single-tube operation, achieving extraction, purification, and detection in one step (the entire process takes only 80 min). This method shows 93% compatibility with ISO-certified RT-qPCR kits, while reducing experimental time and cost by more than 30%, providing a rapid, safe, and low-cost solution for the diagnosis of feline calicivirus.

Claims

1. A kit for detecting and diagnosing feline calicivirus, characterized in that, This includes composite magnetic beads, reverse transcription systems, and quantitative real-time PCR detection systems; The composite magnetic beads are Fe3O4@UiO-66; The reverse transcription system includes reverse transcription primers and reverse transcription reagents, and the nucleotide sequence of the reverse transcription primers is: 5'-ATGGCATGGTTGGGGCAGAC-3'.

2. The reagent kit according to claim 1, characterized in that, The reverse transcription reagent includes an RNase inhibitor, M-MLV reverse transcriptase, and RT buffer.

3. The reagent kit according to claim 1, characterized in that, The quantitative real-time PCR detection system includes amplification primers and reaction reagents.

4. The reagent kit according to claim 3, characterized in that, The amplification primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is 5'-CGTGATCCGAGCAGGAGGTG-3', and the nucleotide sequence of the downstream primer is 5'-ATGGCATGGTTGGGGCAGAC-3'.

5. The reagent kit according to claim 3, characterized in that, The reaction reagents include SYBR qPCR Mix.

6. A method for detecting and diagnosing feline calicivirus using the kit according to any one of claims 1-5, wherein the method is for non-disease diagnostic purposes, characterized in that, Includes the following steps: Step 1: Mix the composite magnetic bead suspension and the reverse transcription primer buffer to obtain a complex of reverse transcription primers and composite magnetic beads; Step 2: Separate the composite magnetic beads from the reaction solution in Step 1 using an external magnetic field, and then mix and incubate the composite magnetic beads with the sample lysis buffer containing viral RNA. Step 3: Add reverse transcription reagent to the mixture from Step 2 to reverse transcribe viral RNA into cDNA on the surface of the composite magnetic beads. Step 4: Separate the composite magnetic beads in the reaction solution of Step 3 by applying an external magnetic field, take the supernatant for fluorescence quantitative PCR detection, and obtain the detection results.

7. The method according to claim 6, characterized in that, In step 1, the concentration of the composite magnetic beads in the composite magnetic bead suspension is 2 mg / mL, the concentration of the reverse transcription primers in the reverse transcription primer buffer is 10 μM, and the volume ratio of the composite magnetic bead suspension to the reverse transcription primer buffer is 2:

1.

8. The method according to claim 6, characterized in that, In step 2, the incubation conditions are 4℃ for 10 minutes.

9. The method according to claim 6, characterized in that, In step 3, the reverse transcription conditions are 50℃ for 15 min and 85℃ for 2 min.

10. The method according to claim 6, characterized in that, In step 4, the quantitative PCR detection system consisted of: 5 μL of SYBR qPCR Mix, 0.2 μL of 10 μM FCV-F, 0.2 μL of 10 μM FCV-R, 2 μL of sds-cDNA, and ddH2O added to 10 μL; the amplification conditions were 95℃ for 30 s, 95℃ for 10 s, and 60℃ for 30 s, for a total of 45 cycles.

Images