PCR (Polymerase Chain Reaction) detection kit for four vertical transmission viruses of waterfowl and application of PCR detection kit

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a PCR (Polymerase Chain Reaction) detection kit for four vertical transmission viruses of waterfowl and application of the PCR detection kit. The kit can rapidly, specifically and simultaneously amplify and diagnose duck hepatitis virus type I (DHV-1), duck Tembusu virus DTMUV, Muscovy duck reovirus MDRV and duck astrovirus DAstV, has high specificity and sensitivity, can accurately and rapidly identify DHV-1, DTMUV, MDRV and DAstV, can complete fluorescent quantitative PCR detection in about 50 minutes, and is simple to operate and practical. The detection period is shortened to 1.5 hours from 6-8 hours of a traditional method, the sensitivity is improved by 10-1000 times, the early diagnosis capability of epidemic diseases is remarkably improved, and the epidemic disease misjudgment rate of a farm is obviously reduced. Abuse of vaccines and drugs is reduced, and sustainable development of the poultry breeding industry is promoted.

Description

Technical Field

[0001] This invention belongs to the technical field of virus detection, specifically relating to PCR detection kits for four vertically transmitted viruses in waterfowl and their applications. Background Technology

[0002] In recent years, the following four pathogens have been prevalent in duck flocks, leading to a decline in the immune function and production performance of ducks, causing serious economic losses to the duck farming industry. Furthermore, they often cause mixed infections through vertical transmission, making disease diagnosis difficult.

[0003] Duck hepatitis virus (DHV) is an acute, highly lethal infectious disease in ducklings, characterized by rapid onset, short course, rapid spread, and high mortality. A typical feature after infection is hemorrhagic inflammation of the liver. The disease has been present for nearly 80 years, since its first discovery in the United States in 1945. It was discovered in my country in the mid-20th century and gradually became widespread throughout the country.

[0004] Duck Tembusu Virus (DTMUV) is a newly emerging acute viral infectious disease affecting waterfowl such as laying ducks, breeding ducks, and geese. The disease primarily occurs during the egg-laying period and is characterized by sudden onset, rapid spread, a significant decrease in feed intake and egg production, and ovarian hemorrhage and degeneration. It has now become a major disease threatening waterfowl farming in my country.

[0005] Muscovy duck reovirus (MDRV) is a viral infectious disease of Muscovy ducks. Clinically, the disease is characterized by leg weakness, numerous white necrotic spots on the surface of the liver and spleen, and enlarged and hemorrhagic kidneys as the main necropsy lesions, and is known as "Muscovy duck hepatic white spot disease" or "Muscovy duck mottled liver disease".

[0006] Duck astrovirus (DAstV) causes viral hepatitis in ducklings. Clinical symptoms after infection typically include opisthotonus and lethargy. The lesions are similar to those seen in duck hepatitis virus infection, primarily manifesting as liver swelling with hemorrhage and congestion, spleen enlargement, and kidney enlargement; a typical characteristic is the presence of urate deposits in the internal organs of some ducklings. This disease is widespread in duck flocks in my country and poses a significant threat to the health of duck farming.

[0007] Currently, traditional methods for detecting DHV, TMUV, MDRV, and DAstV mainly include virus isolation and culture, serological testing, chicken embryo inoculation, hemagglutination inhibition assay, electron microscopy, and conventional RT-PCR. However, these methods are time-consuming, cumbersome, and consume large amounts of samples due to their stepwise detection, low sensitivity in virus isolation, poor specificity in serological testing, and inability to quantify with conventional PCR, thus limiting their application in clinical diagnosis. Multiplex Real-time Quantitative PCR (qPCR) is a molecular biology technique that simultaneously detects multiple target nucleic acids in the same reaction system. By designing multiple pairs of specific primers (or probes), the amplification process is monitored in real time using fluorescence signals. Combined with melting curve analysis (SYBR Green method) or multi-channel fluorescence detection (probe method), simultaneous quantitative detection of multiple pathogens or genes can be achieved. Compared to traditional detection methods, multiplex qPCR offers advantages such as high efficiency, high sensitivity, high specificity, quantitative analysis, and lower cost than traditional single-particle PCR. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide PCR detection kits for four vertically transmitted viruses in waterfowl and their applications.

[0009] The technical content of this invention is as follows: This invention provides a PCR detection primer set for four vertically transmitted viruses in waterfowl, including duck hepatitis virus type I (DHV-1), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and duck astrovirus (DAstV). The PCR detection primer set includes an upstream primer, a downstream primer, and a probe primer; The probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end; The fluorescent reporter group includes one of HEX, CY5, Taxas Red or FAM; The fluorescence quenching group includes BHQ1 or BHQ2.

[0010] The sequences of the upstream primer, downstream primer, and probe primer for PCR detection of duck hepatitis virus type I (DHV-1) are shown in SEQ ID NO. 1~3, respectively; The sequences of the upstream primer, downstream primer, and probe primer for PCR detection of duck Tembusu virus (DTMUV) are shown in SEQ ID NO.4~6, respectively. The sequences of the upstream primer, downstream primer, and probe primer for PCR detection of Muscovy duck reovirus (MDRV) are shown in SEQ ID NO. 7~9, respectively; The sequences of the upstream primer, downstream primer, and probe primer for PCR detection of the duck astrovirus (DAstV) are shown in SEQ ID NO.10~12, respectively.

[0011] This invention also provides the application of the above-mentioned PCR detection primer set for four vertically transmitted viruses in waterfowl in the preparation of reagents for detecting duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus and duck astrovirus; The reagents include PCR detection reagents.

[0012] The present invention also provides a PCR detection kit for the four vertically transmitted viruses in waterfowl, wherein the kit contains the PCR detection primer set described above.

[0013] This invention also provides the application of the PCR detection kits for the above-mentioned four vertically transmitted viruses in waterfowl in the preparation of reagents for detecting duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus and duck astrovirus; When the PCR detection kit is used to detect the virus, the reaction system is as follows: 12.5 μL of 2×Hieff Unicon® qPCRTaqMan Probe Master Mix, primer set, 4 μL of DNA template, and RNaseFreeH2O to a final volume of 25 μL; The primer set includes the following: Primers F (0.2 μL), R (0.2 μL), and P (0.1 μL) for duck hepatitis virus type I (DHV-1) were prepared at concentrations of 20 μM. Primers F (0.25 μL), primer R (0.25 μL), and probe primer P (0.125 μL) for duck Tembusu virus DTMUV were prepared at concentrations of 20 μM. Primers F (0.25 μL), primer R (0.25 μL), and probe primer P (0.125 μL) for Muscovy duck reovirus (MDRV) were prepared at concentrations of 20 μM. Primers F (0.3 μL), R (0.3 μL), and P (0.15 μL) for duck astrovirus (DAstV) were prepared at concentrations of 20 μM. PCR amplification conditions were 95℃ for 20s pre-denaturation; 95℃ for 3s, 55℃ for 40s, for a total of 40 cycles.

[0014] The DNA template was obtained from the RNA of duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus, and duck astrovirus. The PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Code: RR036Q) reverse transcription kit was used. The reaction system was 20 μL. 16 μL of RNA was mixed with 4 μL of RT premix and reacted at 37°C for 15 min. Then, it was quickly placed on ice for 2 min to obtain the reverse transcription product, which was the DNA template.

[0015] This invention also provides a PCR detection method for four vertically transmitted viruses in waterfowl for non-diagnostic purposes, comprising the following steps: 1) Extract RNA from the sample to be tested and reverse transcribe it into cDNA; 2) Using the above cDNA as a template, perform multiplex qPCR amplification using the PCR detection primer set, the kit, or the detection system described above; After the reaction is complete, the amplification curve is used to determine whether the sample tested is positive for duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus, and duck astrovirus. The judgment criteria are as follows: if the sample to be tested shows an amplification curve and Ct≤35, it is judged as positive; if the sample to be tested does not show an amplification curve or Ct value>35, ​​it is judged as negative.

[0016] The beneficial effects of this invention are as follows: This invention relates to a PCR detection kit for four vertically transmitted viruses in waterfowl. This kit can rapidly and specifically amplify and diagnose duck hepatitis virus type I (DHV-1), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and duck astrovirus (DAstV) simultaneously. The optimal concentration ratio has been experimentally verified. Furthermore, this method exhibits high specificity and sensitivity, and can accurately and rapidly identify DHV-1, DTMUV, MDRV, and DAstV. Quantitative real-time PCR detection can be completed in approximately 50 minutes, and the operation is simple and practical.

[0017] This invention addresses the problems of high mixed infection rates of duck hepatitis virus type I (DHV-1), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and duck astrovirus (DAstV) in waterfowl farming, as well as the low efficiency and high cost of traditional detection methods. It proposes a "one-tube four-detection" technology based on multiplex real-time quantitative RT-PCR (RT-qPCR). By integrating bioinformatics target design, probe optimization, and clinical validation, it achieves efficient simultaneous detection of four viruses, reducing the detection cycle from 6-8 hours using traditional methods to 1.5 hours, increasing sensitivity by 10-1000 times, significantly improving early disease diagnosis capabilities, and markedly reducing the misdiagnosis rate of diseases in farms. This also reduces the overuse of vaccines and drugs, contributing to the sustainable development of the poultry farming industry. Attached Figure Description

[0018] Figure 1 The results show the concentrations of each component in the system, as well as the screening results for primers and probes in Example 1. Figure 2 The results of specific detection of DHV-1, DTMUV, MDRV, and DAstV using the detection kit in Example 2 are shown. Curve 1 is the amplification curve of MDRV positive sample, curve 2 is the amplification curve of DHV-1 positive sample, curve 3 is the amplification curve of DAstV positive sample, curve 4 is the amplification curve of DTMUV positive sample, and curves 5-10 are the amplification curves of duck plague virus, Muscovy duck parvovirus, duck adenovirus, duck circovirus, and duck Riemerella anatipestifer, respectively. Figure 3 The results of sensitivity detection of DHV-1, DTMUV, MDRV, and DAstV using the detection kit in Example 3 are as follows. Figure 3 In a, 1~10 represent copies of 3.27×10⁻⁶. 10 -3.27×10 1 Amplification curves using MDRV copies / μL as templates. Figure 3 In b, 1~10 represent copies of 2.51×10⁻⁶. 10 -2.51×10 1 Amplification curves using DHV-1 copies / μL as templates. Figure 3 In c, 1~10 represent copies of 3.19 × 10⁻⁶. 10 -3.19×10 1 Amplification curves using DAstV copies / μL as templates. Figure 3 In d, 1~10 represent copies of 1.06×10⁻¹⁰. 10 -1.06×10 1 Amplification curves using DTMUV copies / μL as templates. Detailed Implementation

[0019] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0020] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0021] Example 1 Primer and probe screening: Based on conserved regions of DHV-1 VP1, DTMUV RdRp, MDRV sigma C, and DAstV ORF1b, multiple sets of primers and probes were designed, such as... Figure 1 The results show the concentrations of each component, primers, and probes. The final primer set selected is as follows: The sequences of the upstream primer, downstream primer and probe primer of duck hepatitis virus type I (DHV-1) are shown in SEQ ID NO. 1~3, respectively. The 5' end of the probe is labeled with the FAM group and the 3' end is labeled with the BHQ1 group. The sequences of the upstream primer, downstream primer and probe primer of duck Tembusu virus (DTMUV) are shown in SEQ ID NO. 4~6, respectively. The 5' end of the probe is labeled with the HEX group and the 3' end is labeled with the BHQ1 group. The sequences of the upstream primer, downstream primer, and probe primer of Muscovy duck reovirus (MDRV) are shown in SEQ ID NO. 7~9, respectively. The 5' end of the probe is labeled with the CY5 group, and the 3' end is labeled with the BHQ2 group. The sequences of the upstream primer, downstream primer, and probe primer of duck astrovirus (DAstV) are shown in SEQ ID NO. 10~12, respectively. The 5' end of the probe is labeled with the Taxas Red group, and the 3' end is labeled with the BHQ2 group.

[0022] Multiple sets of designed primers and probes were used for quantitative real-time PCR amplification. The specific quantitative real-time PCR method is as follows: 1) Extracting RNA from clinical samples The samples used were from a waterfowl breeding farm in Guangdong Province that had been diagnosed with DHV-1, DTMUV, MDRV, and DAstV infections, respectively. After obtaining these pathogens, they were validated: PCR amplification and sequencing were performed using cDNA of DHV-1, DTMUV, MDRV, and DAstV as templates with sequencing primers. The sequencing primer sequences for DHV-1, DTMUV, MDRV, and DAstV are shown in SEQ ID NO. 13~20, respectively, and the resulting protein chain lengths were 726bp, 706bp, 516bp, and 735bp, respectively. The MDRV sequence obtained from the analysis showed 100% homology with the partial sequence of MDRV ZJ99 sigma C (GenBank: AY619690.1) published in NCBI. The DTMUV sequence showed 100% homology with the partial sequence of DTMUV HN2015 3291-3500 (GenBank: MN649261.1). The DAstV sequence showed 100% homology with the partial sequence of DAstV AHK-2022 ORF1b (GenBank: OR134528.1). The DHV-1 sequence showed 99.4% homology with the partial sequence of DHV-1 CP VP1 (GenBank: KF826760.1). Finally, the strains were identified as DHV-1, DTMUV, MDRV, and DAstV.

[0023] 2) Reverse transcription system and process: The PrimeScript™ RT Master Mix (Perfect RealTime) (TaKaRa Code: RR036Q) reverse transcription kit was used. The reaction system was 20 μL. The specific operation was as follows: 16 μL of RNA was mixed with 4 μL of RT premix, reacted at 37℃ for 15 min, and then quickly placed on ice for 2 min. The reverse transcription product was stored at -20℃ as a template for subsequent experiments.

[0024] 3) Quantitative PCR reaction system: Using the Hieff Unicon® qPCR TaqMan Probe Master Mix quantitative PCR kit, the following optimal reaction system was obtained after multiple validations, as detailed in Table 1 below: Table 1 Quantitative PCR reaction system

[0025] Amplification conditions: The above PCR system was placed on a BIO-RAD CFX Opus 96 Real-Time PCR System for amplification reaction. The amplification conditions were: 95℃, pre-denaturation for 20s; 95℃, 3s, 55℃, 40s, for a total of 40 cycles.

[0026] Example 2 Specificity test The optimal primers and probes obtained in Example 1 were used for quantitative real-time PCR amplification. The amplification curves were used to rapidly and accurately identify DHV-1, DTMUV, MDRV, and DAstV. The specific quantitative real-time PCR method is as follows: 1) The nucleic acid extraction process of the samples (DHV-1, DTMUV, MDRV, DAstV, duck plague virus, Muscovy duck parvovirus, duck adenovirus, duck circovirus and duck plague Riemerella) is as described in Example 1.

[0027] 2) The reverse transcription system and process are as described in Example 1.

[0028] 3) The quantitative PCR reaction system is the same as in Example 1.

[0029] 4) Result Interpretation: Samples exhibiting an amplification curve and a CT value less than 35 are considered positive. Specifically, a CT value less than 35 indicates a positive result, while a CT value greater than 35 exceeds the sensitivity of this method and is considered negative. Detection results are shown in Table 2 and... Figure 2 The results showed that, apart from the four amplification curves for DHV-1, DTMUV, MDRV, and DAstV, no other amplification curves were observed, indicating that the detection method has good specificity.

[0030] Table 2 Test results of the samples to be tested

[0031] Example 3 Sensitivity test The sequence fragments amplified and sequenced using the sequencing primers described in Example 1 were ligated into the cloning vector pMD19-T (Dalian Takara Bio Inc.) to prepare standard positive plasmids (pMD19-DHV-1 VP1, pMD19-DTMUV RdRp, pMD19-MDRV sigma C, and pMD19-DAstV ORF1). The concentration of pMD19-DHV-1 VP1 was determined to be 2.51 × 10⁻⁶. 10 The pMD19-DTMUV RdRp concentration was 1.06 × 10⁻⁶ copies / μL. 10 The pMD19-MDRV Sigma C concentration was 3.27 × 10⁻⁶ copies / μL. 10 The copies / μL and pMD19-DAstV ORF1 concentration were 3.19 × 10⁻⁶. 10 copies / μL.

[0032] Four positive plasmids were used as templates by serially diluting them 10-fold with sterile double-distilled water. 2 μL of each dilution was used as template, and the quadruple real-time PCR method described in Example 2 was used for detection. The amplification curves were observed, and the sensitivity was calculated based on the highest dilution with the expected positive curve. The detection results are as follows: Figure 3 As shown, the results indicate that the minimum detection limit for pMD19-DHV-1 VP1 is 25.1 copies / μL, the minimum detection limit for pMD19-DTMUV RdRp is 10.6 copies / μL, the minimum detection limit for pMD19-MDRV Sigma C is 32.7 copies / μL, and the minimum detection limit for pMD19-DAstV ORF1 is 31.9 copies / μL, thus demonstrating that the detection method has good sensitivity.

[0033] This invention presents a quadruple real-time PCR detection primer, probe, and kit for DHV-1, DTMUV, MDRV, and DAstV. The primers and probes exhibit high specificity and sensitivity, and can identify DHV-1, DTMUV, MDRV, and DAstV. They can be used to detect various clinical samples, enabling rapid diagnosis of DHV-1, DTMUV, MDRV, and DAstV. The method is simple and practical. Currently, there is no existing real-time PCR detection method for simultaneously detecting DHV-1, DTMUV, MDRV, and DAstV among the four vertically transmitted viruses in waterfowl. Therefore, establishing a real-time PCR detection kit for these four RNA viruses in waterfowl is of great significance for timely monitoring and disease control of DHV-1, DTMUV, MDRV, and DAstV.

Claims

1. A primer set for PCR detection of four vertically transmitted viruses in waterfowl, characterized by, The four vertically transmitted viruses of waterfowl include duck hepatitis virus type I (DHV-1), duck tembusu virus (DTMUV), muscovy duck reovirus (MDRV) and duck astrovirus (DAstV); The PCR detection primer set comprises an upstream primer, a downstream primer and a probe primer; The sequences of the PCR detection upstream primer, the downstream primer and the probe primer of the duck hepatitis virus type I (DHV-1) are shown in SEQ ID NO. 1-3, respectively; The sequences of the PCR detection upstream primer, the downstream primer and the probe primer of the duck tembusu virus (DTMUV) are shown in SEQ ID NO. 4-6, respectively; The sequences of the PCR detection upstream primer, the downstream primer and the probe primer of the muscovy duck reovirus (MDRV) are shown in SEQ ID NO. 7-9, respectively; The sequences of the PCR detection upstream primer, the downstream primer and the probe primer of the duck astrovirus (DAstV) are shown in SEQ ID NO. 10-12, respectively.

2. The primer set for PCR detection of four vertically transmitted viruses in waterfowl according to claim 1, wherein, The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; The fluorescent reporter group comprises one of HEX, CY5, Taxas Red or FAM; The fluorescent quencher group comprises BHQ1 or BHQ2.

3. The use of the PCR detection primer set for four kinds of vertically transmitted viruses of waterfowl according to claim 1 or 2 in the preparation of reagents for detecting duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus and duck astrovirus, characterized in that, The reagent comprises a PCR detection reagent.

4. A PCR detection kit for four vertically transmitted viruses of waterfowl, characterized by, The kit contains the PCR detection primer set of claim 1 or 2.

5. Use of the PCR detection kit of the four vertically transmitted viruses of waterfowl in claim 4 in the preparation of a reagent for detecting duck hepatitis virus type I, duck tembusu virus, muscovy duck reovirus and duck astrovirus.

6. Use according to claim 5, characterized in that, When the PCR detection kit is used to detect viruses, the reaction system is: 2xHieff qPCR TaqMan Probe Master Mix 12.5 μL, primer set, DNA template 4 μL, RNase Free H2O, and the rest is made up to 25 μL.

7. Use according to claim 6, characterized in that, In the primer set, the primer F of duck hepatitis virus type I (DHV-1) is 0.2 μL, the primer R is 0.2 μL, and the probe primer P is 0.1 μL, and the concentrations are 20 μM, respectively; The primer F of duck tembusu virus (DTMUV) is 0.25 μL, the primer R is 0.25 μL, and the probe primer P is 0.125 μL, and the concentrations are 20 μM, respectively; The primer F of muscovy duck reovirus (MDRV) is 0.25 μL, the primer R is 0.25 μL, and the probe primer P is 0.125 μL, and the concentrations are 20 μM, respectively; The primer F of duck astrovirus (DAstV) is 0.3 μL, the primer R is 0.3 μL, and the probe primer P is 0.15 μL, and the concentrations are 20 μM, respectively.

8. Use according to claim 5, characterized in that, When the PCR detection kit is used to detect viruses, the PCR amplification conditions are 95℃, pre-denaturation for 20s; 95℃, 3s, 55℃, 40s, for a total of 40 cycles.

9. Use according to claim 6, characterized in that, The DNA template is obtained by reverse transcription of RNA of duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus and duck astrovirus using PrimeScript™ RT Master Mix reverse transcription kit, and the reaction system is 20 μL system, 16 μL RNA is mixed with 4 μL RT premix, 37°C reaction for 15 min, then quickly placed on ice for 2 min, and the reverse transcription product is the DNA template.

10. A PCR method for the detection of four vertically transmitted viruses in waterfowl for non-diagnostic purposes, characterized by, The method comprises the following steps: 1) extracting RNA of the sample to be detected and reverse transcribing into cDNA; 2) using the cDNA as the template, using the PCR detection primer group of claim 1 or 2, or the kit of claim 4, or the PCR reaction system for multiplex qPCR amplification; After the reaction is completed, whether the duck hepatitis virus type I, duck Tembusu virus, Muscovy duck reovirus and duck astrovirus in the sample to be detected are positive is determined according to the amplification curve; The determination standard is as follows: if the amplification curve appears in the detection of the sample to be detected and Ct≤35, it is determined to be positive; if the amplification curve is not detected or Ct>35, it is determined to be negative.

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