Composition for detecting multi-host Kubu virus as well as kit and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a composition for detecting a multi-homed Kubu virus as well as a kit and application thereof. The invention discloses a reagent which comprises a primer pair and / or a probe. The reagent provided by the invention can be used for detecting the Kubu virus, can be used for simultaneously and rapidly detecting and identifying the Kubu virus in different host samples of livestock (pigs, cattle and sheep), companion animals (dogs and cats), wild animals (masked civet and rats) and the like, has extremely high detection sensitivity and specificity, and is accurate and reliable in detection result.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to compositions, kits, and applications for detecting multi-host Kubvirus. Background Technology

[0002] Kobuvirus belongs to the Picornaviridae family (Periviruses). Picornaviridae Cobuviruses belong to the genus Cobuvirus, which are single-stranded positive-sense RNA viruses with a wide host spectrum, infecting various animals including humans, pigs, cattle, dogs, and rodents (Table 1). Clinical manifestations vary among different hosts: in humans, infection can cause acute gastroenteritis (more common in infants and young children), manifesting as diarrhea and vomiting; in pigs, infection can lead to diarrhea, growth retardation, and in severe cases, death in piglets; cattle and dogs may also exhibit gastrointestinal symptoms or latent infection, posing a potential threat to public health, livestock production, and the health of companion animals. Currently, the main methods for detecting Kobuvirus include virus isolation and culture, serological testing (such as indirect ELISA), and conventional RT-PCR testing. However, existing technologies have significant limitations: virus isolation and culture rely on cell lines (such as Vero cells and PK-15 cells), which are cumbersome, time-consuming (3-5 days), and have low isolation efficiency, making it difficult to meet the needs of rapid detection; serological testing relies on specific antibodies, is easily affected by cross-reactions (with other small RNA viruses), and cannot distinguish between acute and past infections, resulting in insufficient sensitivity; conventional RT-PCR testing, although faster, has poor specificity, is prone to false positives, and can only perform qualitative detection, unable to accurately quantify viral load. It is also difficult to adapt to detection scenarios where there are large differences in viral genotypes and a wide range of viral loads in multi-host samples (such as low viral load in latently infected wild animal samples and high viral load in diseased animal samples). Furthermore, existing Kobuvirus detection methods are mostly designed for single hosts (such as humans or pigs only), lacking a universal detection technology that can simultaneously cover viral strains from multiple host sources. This limits efforts in cross-host transmission monitoring and epidemic tracing. Therefore, developing a highly specific, sensitive, rapid, and quantitative Kobuvirus detection method that is adaptable to multiple hosts and genotypes is of significant practical importance for the prevention, monitoring, and research of this virus.

[0003] Table 1. Classification and host animals of Kubuvir Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing Kobuvirus detection methods and provide a multi-host Kobuvirus qPCR detection method to achieve rapid, sensitive, and specific quantitative detection of Kobuvirus in different hosts and with different genotypes, including livestock (pigs, cattle, sheep), companion animals (dogs, cats), and wild animals (civets, rodents). This method meets the application needs of multiple scenarios such as clinical practice, animal husbandry, and disease control, and provides technical support for cross-host transmission monitoring.

[0005] The first aspect of the present invention is to provide a reagent.

[0006] A second aspect of the present invention is to provide a reagent kit.

[0007] The object of a third aspect of the present invention is to provide the application of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention.

[0008] The fourth aspect of this invention aims to provide a method for detecting Kubuvir.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a reagent comprising primer pairs and / or probes; The nucleotide sequences of the primer pair are shown below: Kov-F: 5'-TCCACGATHTATGAAGTCAC-3', or the complementary sequence of this sequence; Kov-R: 5'-AATCTCCGTCHCGCADCCACAT-3', or the complementary sequence of this sequence; The nucleotide sequence of the probe is shown below: Kov-P: 5'-TCCTCAAGCGCTGGTTTGT-3', or the complementary sequence of this sequence.

[0010] In some embodiments of the present invention, the probe sequence is labeled with a fluorescent group and a quenching group at both ends.

[0011] In some embodiments of the present invention, the fluorescent group is at least one of FAM, Hex, VIC, TAMRA, ROX, Texas-Red and CY5.

[0012] In some embodiments of the present invention, the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0013] In some embodiments of the present invention, the fluorescent group is attached to the 5' end of the probe.

[0014] In some embodiments of the present invention, the quenching group is attached to the 3' end of the probe.

[0015] In some preferred embodiments of the present invention, the 5' end of the Kov-P is connected to the fluorescent group CY5, and the 3' end is connected to the quenching group BHQ2.

[0016] A second aspect of the present invention provides a reagent kit comprising the reagent of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the kit further includes: PCR buffer, DNA polymerase, dNTPs, and Mg. 2+ At least one of them.

[0018] In some embodiments of the present invention, the kit further comprises reverse transcription primers and reverse transcriptase for reverse transcribing RNA into cDNA.

[0019] In some embodiments of the present invention, the reverse transcription primer is Oligo(dT) or a random primer.

[0020] In some embodiments of the present invention, the kit further comprises: a positive control sample containing Kub virus and a negative control sample not containing Kub virus.

[0021] In some embodiments of the present invention, the kit is used for at least one of a1)-a4): a1) Identification of Kubuvir; a2) Identify whether the virus to be tested is Kubuvir; a3) Identification of Kubuvir; a4) Detect whether the sample to be tested contains Kubuvir.

[0022] A third aspect of the present invention provides the use of the reagent of the first aspect of the present invention or the kit of the second aspect of the present invention in any one of (1)-(6); (1) Identification of Kubuvir; (2) Preparation of products for identifying Kubuvir; (3) Detect whether the sample to be tested is Kubuvir; (4) Prepare products for testing or assisting in the testing of whether the sample to be tested is Kubuvir; (5) Detect whether the sample to be tested is infected with Kubuvir; (6) Prepare products for detecting or assisting in the detection of whether a sample to be tested is infected with Kubuqi virus; The above applications are used for the diagnosis and treatment of non-disease conditions.

[0023] A fourth aspect of the present invention provides a method for detecting Kubuvir, comprising the steps of using reagents from the first aspect of the present invention and / or kits from the second aspect of the present invention for detection, said method being used for non-disease diagnosis and treatment.

[0024] In some embodiments of the present invention, the detection method includes the following steps: RNA was extracted from the sample to be tested and reverse transcribed into cDNA; Using cDNA as a template, PCR amplification was performed with the reagent or kit described above, and fluorescence signals were collected. The presence of Kubuvir in a sample is determined based on the fluorescence signal.

[0025] In some embodiments of the present invention, the final concentrations of Kov-F and Kov-R in the PCR amplification reaction system are 100-500 nM, such as any value of 100, 150, 200, 250, 300, 350, 400, 450 or 500 nM or any range formed by both.

[0026] In some embodiments of the present invention, the final concentration of Kov-P in the PCR amplification reaction system is 50-200 nM, such as any value of 50, 100, 150 or 200 nM or a range formed by any two of them.

[0027] In some embodiments of the present invention, the PCR amplification reaction program is as follows: 35-40℃, 2-3 min; 94-96℃, 3-7 min; 90-95℃, 8-10 s; 50-65℃, 25-35 s; 40-50 cycles.

[0028] In some embodiments of the present invention, the PCR amplification reaction program is as follows: 35-37℃, 2-3 min; 94-96℃, 4-6 min; 94-95℃, 8-10 s; 50-60℃, 30-35 s; 40-45 cycles.

[0029] In some preferred embodiments of the present invention, the annealing temperature in the PCR amplification reaction procedure is 55-60°C, such as any value of 55, 56, 57, 58, 59 or 60°C or a range formed by both.

[0030] In some embodiments of the present invention, the result determination method of the detection method is as follows: based on the experimental validity, that is, the negative control has no Ct value and no regular amplification curve, and the positive control Ct value should be less than 36.0, if the Ct value of the test sample is less than 36.0 and a typical amplification curve appears, it is judged as positive; if the Ct value of the test sample is greater than 36.0 and the amplification curve is consistent, it is judged as suspicious and needs to be retested. If the test result is consistent with the previous result, it is judged as positive; if it is inconsistent, it is judged as negative; if the test sample has no Ct value or is greater than 36.0 and has no regular amplification curve, it is judged as negative.

[0031] The beneficial effects of this invention are: The reagents provided by this invention can be used to detect Kubuvir, and can simultaneously and rapidly detect and identify Kubuvir in different host samples such as livestock (pigs, cattle, sheep), companion animals (dogs, cats), and wild animals (civets, rodents), and have extremely high detection sensitivity and specificity, with accurate and reliable detection results.

[0032] This invention provides a detection method for multi-host Coobuvirus, applicable to rapid screening, specific identification, and quantitative analysis of Coobuvirus in samples from different hosts such as livestock, companion animals, and wild animals. It features high detection sensitivity (detection limit of 2.8 copies / μL), strong specificity, good repeatability, low cost, and can simultaneously detect a large number of samples. It also shows no cross-reactivity with other viruses, making it ideal for detecting large numbers of clinical samples and monitoring epidemics. It can be widely used in clinical diagnosis, animal husbandry and veterinary quarantine, and wildlife disease monitoring. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The amplification curve and standard curve are shown for detecting plasmid standards using the detection method in Example 3; where A is the amplification curve and B is the standard curve.

[0034] Figure 2 The results show the sensitivity test results of the detection method in Example 3.

[0035] Figure 3 This is the specific detection result of the detection method in Example 3. Detailed Implementation

[0036] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0037] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] Example 1: Target selection, primer and probe screening and optimization The complete genome sequences of Kobuvirus from different host sources (swine, cattle, sheep, dogs, cats, civets, and rodents) were obtained from the website of the National Center for Biotechnology Information (NCBI). Conserved sequences were screened using Mega 11.0, and the highly conserved 3D polymerase gene sequence of Kobuvirus was finally used as the target. Specific primers and fluorescent probes were designed using Primer 6.0 and Oliogo7. The nucleotide sequences are shown in Table 2 below.

[0040] Table 2 Primer and probe sequences

[0041] Kov-P is labeled with carboxyfluorescein CY5 at its 5' end and with quencher group BHQ2 at its 3' end.

[0042] Example 2: Construction of plasmid standards To ensure uniform plasmid concentration during subsequent Kubuvir optimization, the conserved sequence of Kubuvir was synthesized into the PUC57 plasmid, yielding PUC57-Kov. The plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. According to the formula (concentration ng / μL × 6.02 × 10⁻⁶), the concentration was determined to be 9.5%. 23 ×10 -9 Calculate the copy number of the plasmid standard using (DNA length × 660) and dilute it to the appropriate copy number.

[0043] Example 3: Establishment and optimization of RT-qPCR detection method An RT-qPCR detection method for detecting multi-host Kubvirus includes the following steps: (1) Obtain the RNA of the sample to be tested and reverse transcribe it into cDNA; (2) Using the nucleic acid in step (1) as a template, RT-qPCR was performed using the primer and probe combination designed and screened in Example 1. The RT-qPCR amplification reaction system was as follows: 20 μL system consisted of 2 μL template, 10 μL probe reaction solution 2×AceQUniversal U+Probe Master Mix V2 (Q113-02, purchased from Nanjing Novizan Biotechnology Co., Ltd.), 200 nM Kov-F / R (final concentration), 100 nM Kov-P (final concentration), and ddH2O was used to make up to 20 μL. The reaction program was as follows: digestion at 37℃ for 2 min, pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, 45 cycles. (3) Amplification was performed using the above reaction procedure and system to obtain amplification kinetic curves. The standard linear regression equation (standard curve) was derived by using the common logarithm (lgC) of the initial copy number of the standard (prepared in Example 2) as the x-axis and the cycle number threshold (Ct value) as the y-axis, and its sensitivity test data were obtained.

[0044] Result Interpretation: Based on the assumption that the experiment is valid (negative control has no Ct value and no regular amplification curve), and the positive control has a Ct value less than 36.0, if the test sample has a Ct value less than 36.0 and shows a typical amplification curve, it is considered positive. If the test sample has a Ct value greater than 36.0 and the amplification curve is consistent, it is considered suspicious and requires retesting. If the test result is consistent with the previous result, it is considered positive; otherwise, it is considered negative. If the test sample has no Ct value or a Ct value greater than 36.0 and has no regular amplification curve, it is considered negative.

[0045] The reaction conditions for the above RT-qPCR detection method were further optimized as follows: (1) Optimization of annealing temperature The annealing temperature (Tm) for RT-qPCR of Kubuvir was optimized using a matrix method. The primer annealing temperatures were divided into three groups: 50℃, 55℃, and 60℃. Each group underwent gradient amplification (2.8 × 10⁻⁶). 6 -2.8×10 2 (Copies / μL), with a negative control included in each group. The reaction system followed the manufacturer's recommended system, as shown in Table 3 below. The reaction conditions were: digestion at 37℃ for 2 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s; annealing at 50℃ / 55℃ / 60℃ for 30 s, repeated 40 times. After the reaction, the amplification curve data were analyzed, a standard curve was plotted, and the amplification efficiency E was calculated. Then, based on a comprehensive consideration of amplification efficiency, fluorescence intensity, and other factors, the optimal primer annealing temperature was finally selected.

[0046] The results of annealing temperature optimization are shown in Table 4. The results show that the amplification efficiency is highest at 60℃.

[0047] Table 3. Reaction system under optimized annealing temperature

[0048] Table 4 Results of Annealing Temperature Optimization

[0049] (2) Primer concentration optimization Primer concentrations were optimized using a matrix method. Kubuvir RT-qPCR primers were diluted to 10 μM, with final primer concentrations ranging from 100 to 500 nM. Ten concentration gradients were set, and the plasmid concentration was 2.8 × 10⁻⁶. 6 -2.8×10 2 Amplification was performed using copies / μL, with RNase-free water as a negative control. The optimal primer concentration for Kubuvir was optimized, a standard curve was plotted, and amplification efficiency was calculated. The best primer concentration was selected from different primer concentration combinations.

[0050] The results of primer concentration optimization are shown in Table 5. The results show that the optimal combination of upstream and downstream primers for Kubuvir is 250 nM.

[0051] Table 5 Primer optimization results

[0052] (3) Probe concentration optimization The probe concentration was optimized using a matrix method. The Kubuvir RT-qPCR probe was diluted to 10 μM, with final probe concentrations ranging from 50 to 200 nM, for a total of four concentration gradients. The plasmid concentration was 2.8 × 10⁻⁶. 6 -2.8×10 2 Amplification was performed using copies / μL, with RNase-free water as a negative control. The optimal probe concentration for Kubuvir was optimized, a standard curve was plotted, and the amplification efficiency was calculated.

[0053] The results of probe concentration optimization are shown in Table 6. The results show that the optimal concentration of the Kubuvir probe is 150 nM.

[0054] Table 6 Probe optimization results

[0055] Based on the above system optimization results, the final reaction system (20 μL) for the RT-qPCR detection method is as follows: 2 μL template, 10 μL probe reaction solution 2×AceQ Universal U+Probe Master Mix V2 (Q113-02, purchased from Nanjing Novizan Biotechnology Co., Ltd.), Kov-F / R 250 nM, Kov-P 150 nM, and ddH2O to make up to 20 μL; the reaction program is as follows: digestion at 37℃ for 2 min, pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 45 cycles.

[0056] Example 4: Establishment of Standard Curve Using plasmid standard PUC57 Using Kov (Example 2) as a template, it was serially diluted 10-fold to obtain: 2.8 × 10⁻⁶ 6 -2.8×10 2 copies / μL, then aliquoted and Store at 20°C; use the optimized detection method from Example 3 to test diluted PUC57. The Kov plasmid was amplified to obtain the template copy number and cycle number (Ct value). After the detection was completed, the concentration lg value (X-axis) of each standard was plotted against its corresponding Ct value (Y-axis) to create a standard curve.

[0057] The obtained template copy number versus cycle number (Ct value) amplification curve is shown below. Figure 1 As shown; after the test, plot the standard concentration lg (copies / μL) value (X-axis) against its corresponding cycle number Ct value (Y-axis) to create a standard curve (e.g. Figure 1 (As shown); Standard curve equation: y = -3.481x + 39.101, R 2 =0.9963. The standard curve equation shows that different concentration gradients are linearly correlated with the Ct value, and Rt... 2 A value greater than 0.99 indicates a good correlation.

[0058] Example 5 Sensitivity Test With 2.8×10 6 -2.8×10 0 Quantitative PCR standard plasmid PUC57 was serially diluted (10-fold) in copies / μL. Using Kov (Example 2) as a template, RT-qPCR detection was performed using the detection method optimized in Example 3.

[0059] The results are as follows Figure 2As shown, the detection limit for Kubuvir is 2.8 copies / μL, corresponding to a cycle number of 35.80; this demonstrates that the detection method established in Example 3 has good sensitivity.

[0060] Example 6 Specificity Detection Nucleic acid was extracted from vaccines or clinical samples of other respiratory viruses, such as rotavirus, porcine epidemic diarrhea virus, bovine viral diarrhea virus, bovine enterovirus, canine parvovirus, and canine distemper virus (all preserved in our laboratory), using DNA or cDNA as templates, PUC57. Kov was used as a positive control and ddH2O as a negative control. RT-qPCR detection was performed according to the optimized reaction system and conditions in Example 3.

[0061] result Figure 3 As shown, only PUC57 The Cov plasmid control showed an "S"-shaped amplification curve with high fluorescence intensity and no non-specific amplification. Other respiratory viruses and the negative control showed no amplification curves and no Ct value was observed, proving that the RT-qPCR detection method constructed in Example 3 has high specificity, with a detection specificity of 100%.

[0062] Example 7 Repeatability Test Standard plasmid PUC57 Kov (Example 2) was serially diluted 10-fold to: 2.8 × 10⁻⁶ 6 copies / μL, 2.8×10 5 copies / μL, 2.8×10 4 copies / μL, 2.8×10 3 copies / μL, 2.8×10 2 The samples were collected in copies / μL and tested using the optimized detection method from Example 3. RT-qPCR was performed with three replicates each time, for a total of three cycles. The mean cycle number, standard deviation, and coefficient of variation were calculated.

[0063] The results are shown in Table 7. The coefficients of variation within and between groups are all less than 2%, which proves that the detection method established in Example 3 has good repeatability.

[0064] Table 7. Repeatability test results of the detection method

[0065] Example 8: Detection of Clinical Samples (1) Sample information and nucleic acid extraction: ① Civet cat tissue: 105 samples including liver, intestine and stomach from 35 civet cats collected from a breeding farm in Guangdong Province; ② Pig feces: 80 pig feces samples collected from different pig farms in Guangdong Province; ③ Dog feces: 50 dog feces samples collected from a veterinary hospital; The above samples were stored in the laboratory at -20℃. After the above samples were prepared into suspensions with PBS, viral RNA was extracted according to the operation method of the viral RNA / DNA extraction kit (R4173-02, purchased from Guangzhou Meiji Biotechnology Co., Ltd.), and reverse transcribed into cDNA using the full-length cDNA one-strand synthesis kit (R312-01 / 02, purchased from Nanjing Novizan Biotechnology Co., Ltd.) for later use.

[0066] (2) Comparison method: The test samples were tested according to the comparison method in Table 8 and the detection method in Example 3.

[0067] Table 8 Comparison Methods

[0068] Detection Results: As shown in Table 9, for civet cobuvirus and canine cobuvirus, the detection method of Example 3 of this invention showed a 100% concordance rate with existing mature methods (i.e., the methods in Table 8). Positive samples were verified by sequencing to be the target virus, demonstrating that the detection method of Example 3 has extremely high detection accuracy for cobuviruses from these two host sources. For swine cobuvirus, the detection method of Example 3 detected two positive samples that were not identified by the comparison method. Upon review and sequencing, these two samples were confirmed to be positive for cobuvirus, suggesting that the detection method of Example 3 may have higher detection sensitivity and can capture low-viral-load infection samples missed by the comparison method. The above results indicate that the detection method of Example 3 can rapidly, accurately, and reliably detect and identify civet cobuvirus, swine cobuvirus, and canine cobuvirus in clinical samples.

[0069] Table 9 Clinical Sample Test Results

[0070] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent comprising a primer pair and / or a probe; The nucleotide sequences of the primer pair are shown below: Kov-F: 5'-TCCACGATHTATGAAGTCAC-3', or the complementary sequence of this sequence; Kov-R: 5'-AATCTCCGTCHCGCADCCACAT-3', or the complementary sequence of this sequence; The nucleotide sequence of the probe is shown below: Kov-P: 5'-TCCTCAAGCGCTGGTTTGT-3', or the complementary sequence of this sequence.

2. The reagent according to claim 1, characterized in that, The probe is labeled with a fluorescent group and a quenching group at both ends of its sequence.

3. The reagent according to claim 2, characterized in that, The fluorescent group is at least one of FAM, Hex, VIC, TAMRA, ROX, Texas-Red and CY5; and / or, the quenching group is at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

4. A reagent kit, characterized in that, The kit comprises the reagent according to any one of claims 1-3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes: PCR buffer, DNA polymerase, dNTPs, and Mg. 2+ At least one of them.

6. The use of the reagent of any one of claims 1-3 or the kit of claim 4 or 5 in any one of (1)-(6); (1) Identification of Kubuvir; (2) Preparation of products for identifying Kubuvir; (3) Detect whether the sample to be tested is Kubuvir; (4) Prepare products for testing or assisting in the testing of whether the sample to be tested is Kubuvir; (5) Detect whether the sample to be tested is infected with Kubuvir; (6) Prepare products for detecting or assisting in the detection of whether a sample to be tested is infected with Kubuqi virus; The above applications are used for the diagnosis and treatment of non-disease conditions.

7. A method for detecting Kubuvir, comprising the step of using the reagent of any one of claims 1-3 and / or the kit of claim 4 or 5, the method being used for non-disease diagnosis and treatment.

8. The detection method according to claim 7, characterized in that, The detection method includes the following steps: RNA was extracted from the sample to be tested and reverse transcribed into cDNA; Using cDNA as a template, PCR amplification was performed with the reagent or kit described above, and fluorescence signals were collected. The presence of Kubuvir in a sample is determined based on the fluorescence signal.

9. The detection method according to claim 8, characterized in that, In the PCR amplification reaction system, the final concentrations of Kov-F and Kov-R are 100-500 nM; and / or, the final concentration of Kov-P is 50-200 nM.

10. The detection method according to claim 8 or 9, characterized in that, The PCR amplification reaction program is as follows: 35-40℃, 2-3 min; 94-96℃, 3-7 min; 90-95℃, 8-10 s; 50-65℃, 25-35 s; 40-50 cycles.

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