Primers and detection method for dual real-time fluorescence quantitative PCR detection of duck hepatitis A virus type 1 and duck astrovirus type 3

Abstract

The present invention relates to the field of viral molecular biology detection by establishing a dual real-time fluorescence quantitative PCR primer set and kit for duck hepatitis A virus type 1 and duck astrovirus type 3. This method can rapidly detect duck hepatitis A virus type 1 and duck astrovirus type 3. The method is simple and rapid, with a single sample detection time of 90 minutes and a sensitivity of up to 10 copies / μl. Simultaneous detection is possible for larger sample sizes, and the method exhibits excellent sensitivity, repeatability, specificity, and stability, enabling rapid and accurate detection of duck hepatitis A virus type 1 and duck astrovirus type 3.

Description

Technical Field

[0001] The present invention relates to the field of virus molecular biology detection, and in particular to primers and a detection method for a multiplex SYBR Green I real-time fluorescence quantitative PCR detection method for DHAV-1 and DAstV-3. Background Art

[0002] Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease caused by a virus that infects ducklings. It is characterized by rapid onset, rapid spread, a short course, and a high mortality rate. The disease first occurred in the United States in 1950, and subsequent prevalence has been reported in countries such as the United Kingdom, Canada, Germany, Italy, India, France, Russia, Hungary, and Japan. DVH was previously classified into three serotypes: serotype 1, serotype 2, and serotype 3. Serotype 1 belongs to the duck hepatitis A virus (DHAV) family, genus Avihepatovirus, and family Picornaviridae. DHAV is currently classified into three genotypes: type 1, type 2, and type 3. Duck hepatitis virus serotype 2 belongs to the family Avastroviridae and the genus Avastrovirus, belonging to duck astrovirus 1 (DAstV-1). Duck hepatitis virus serotype 3 is currently classified as duck astrovirus 2 (DAstV-2) in the family Avastroviridae and the genus Avastrovirus. Subsequently, Chinese researchers discovered duck astrovirus 3 (DAstV-3) and duck astrovirus 4 (DAstV-4) in 2014-2015. This suggests that duck astrovirus infection in ducks also manifests as duck viral hepatitis. Its symptoms are extremely similar to those caused by duck hepatitis A virus, making them difficult to distinguish.

[0003] Duck hepatitis, caused by duck viral hepatitis, develops rapidly, spreads rapidly, has a short course, and has a high mortality rate. Clinical symptoms typically include opisthotonos, ataxia, lethargy, slumping to one side, anorexia, and head-to-the-ground contact. Pathological changes include hepatomegaly and hemorrhagic macules. DHAV-1 and DAstV-3 are widely prevalent in China, and their clinical differentiation is difficult through visual observation, making prevention and control extremely difficult and significantly impacting the duck farming industry.

[0004] At present, there is no clinical method for detecting mixed infection of DHAV-1 and DAstV-3. SYBR Green I real-time fluorescence quantitative polymerase chain reaction technology is a reaction technology that is implemented on the basis of traditional polymerase chain reaction technology (PCR) by using specific primers added and a real-time fluorescence quantitative PCR instrument. Fluorescence quantitative PCR technology has the advantages of high specificity, strong sensitivity, and good repeatability. It can complete the reaction within 2 hours and has intuitive reaction results. Dual real-time fluorescence quantitative PCR technology is a method for quickly diagnosing and distinguishing two viruses based on the different Tm values ​​caused by the difference in the GC content of the fragments amplified by two sets of primers on the basis of single real-time fluorescence quantitative PCR technology.

[0005] The duplex SYBR Green I-based real-time PCR assay developed in this invention is a new technology that enables efficient in vitro amplification of trace amounts of nucleic acids. It can achieve efficient amplification of target fragments in a short period of time. The entire reaction is simple and rapid, with a high detection rate and good reproducibility. It is suitable for use with large sample sizes and has strong potential for widespread application. The development of this invention can fill a gap in the relevant field. Summary of the Invention

[0006] The present invention aims to provide a dual real-time fluorescence quantitative PCR detection primer and method for duck hepatitis A virus type 1 and duck astrovirus type 3. The difference in GC content of the target fragments amplified by the two sets of primers leads to a difference in Tm value. After the completion of the SYBR Green I real-time fluorescence quantitative PCR reaction, combined with the difference in melting temperature, rapid differential diagnosis and infection degree analysis of duck hepatitis A virus type 1 and duck astrovirus type 3 can be performed. The method is simple and rapid, and its sensitivity can reach 10 1 The copy number / ul can be used for simultaneous detection of larger sample amounts, with good sensitivity, repeatability, specificity and stability, and can achieve the purpose of rapid and accurate detection of duck hepatitis A virus type 1 and duck astrovirus type 3 at the same time.

[0007] The present invention is achieved through the following technical solutions:

[0008] The dual real-time fluorescence quantitative PCR detection primers and method for detecting duck hepatitis A virus type 1 and duck astrovirus type 3 are designed based on the VP0 gene of duck hepatitis A virus type 1 and the ORF1b gene of duck astrovirus type 3. Among them, the specific primer sequence of hepatitis A virus type 1 is:

[0009] Upstream primer F1: AACTACACAGACAATACT

[0010] Downstream primer R1: CAGGCATAATAATCATCAT

[0011] Among them, the specific primer sequence of duck astrovirus type 3 is:

[0012] Upstream primer F2: ACGACAGTAGATAACAAC

[0013] Downstream primer R2: GTCATCACCATAACAGAT

[0014] The present invention also provides a double real-time fluorescence quantitative PCR detection method for detecting duck hepatitis A virus type 1 and duck astrovirus type 3 using the specific primers mentioned above.

[0015] The amplification conditions of the dual real-time fluorescence quantitative PCR detection method are as follows: the reaction system is 20ul, SuperReal PreMix Plus 10ul, duck hepatitis A virus type 1 upstream primer, downstream primer 0.2ul each, duck astrovirus type 3 upstream primer, downstream primer 0.4ul each, DNA template 1ul each, and sterile deionized water is added to the final volume of 20ul. TM Amplification reaction was performed on a Real-Time System machine, and the reaction program conditions were set as follows: 95°C, 15 min pre-denaturation; 95°C 10 s, 60°C 30 s, for a total of 40 cycles; after the reaction was completed, a melting curve was generated.

[0016] The advantages of the present invention are as follows:

[0017] (1) High sensitivity, dual real-time fluorescence quantitative PCR detection technology, using the primers provided by the present invention to detect samples, the sensitivity can reach 10 1 The copy number / ul is 100 times more sensitive than the conventional PCR method.

[0018] (2) Good specificity. In this experiment, duck Tembusu virus, new goose parvovirus, duck circovirus, and Newcastle disease virus were detected simultaneously. This method does not react with other common infectious diseases of ducks, and this method has good applicability.

[0019] (3) The operation is simple and the detection time is short. The detection can be completed within 1 hour and 30 minutes at the fastest.

[0020] (4) The results are reliable. In the present invention, a fluorescence quantitative instrument is used to automatically generate the results, thereby reducing the contamination caused by human operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Establishment of DHAV-1 standard curve and DHAV-1 sensitivity experiment.

[0022] Figure 2 Establishment of DAstV-3 standard curve and DAstV-3 sensitivity experiment.

[0023] Figure 3 Establishment of DHAV-1 dissolution curve.

[0024] Figure 4 Establishment of DAstV-3 dissolution curve.

[0025] Figure 5 Establishment of dual dissolution curves for DHAV-1 and DAstV-3.

[0026] Figure 6 Duplex real-time fluorescence quantitative PCR specificity experiment. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below through specific embodiments. The examples in this description are only used to illustrate the present invention and are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, some modifications or replacements may be made to the details and forms of the technical solution of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0028] Example 1:

[0029] Validation experiment of the dual real-time fluorescence quantitative PCR method for detecting duck hepatitis A virus type 1 and duck astrovirus type 3 1. Design of primers for dual real-time fluorescence quantitative PCR experiment

[0030] Specific primer sequences for the VP0 gene of duck hepatitis A virus type 1 and the ORF1b gene of duck astrovirus type 3 were designed to simultaneously detect duck hepatitis A virus type 1 and duck astrovirus type 3:

[0031] Forward primer of hepatitis A virus type 1: AACTACACAGACAATACT. Downstream primer: CAGGCATAATAATCATCAT.

[0032] For duck astrovirus type 3, the upstream primer is ACGACAGTAGATAACAAC. The downstream primer is GTCATCACCATAACAGAT.

[0033] The upstream and downstream primers were synthesized by Universal Corporation.

[0034] 2. Virus strain

[0035] The duck hepatitis A virus type 1 and duck astrovirus type 3 used in the experiment were identified and preserved in our laboratory.

[0036] 3. Extraction of viral nucleic acid

[0037] The samples were collected to extract the viral genome according to the instructions of the Tiangen Genome Kit (Tiangen Biotechnology Co., Ltd.), and after the extraction was completed, they were stored at -20℃ for future use.

[0038] 4. Preparation of positive samples

[0039] Using extracted duck hepatitis A virus type 1 and duck astrovirus type 3 RNA as templates, amplification was performed using the designed specific primers. The target amplified products were purified and recovered using a Tiangen Gum recovery kit after electrophoresis. The pMD-19T vector was then used for ligation, transformation, colony PCR identification, and sequencing analysis. Positive samples were extracted and purified in small quantities and stored at -20°C until use. The positive standards were designated pMD-19T-DHAV-1 and pMD-19T-DAstV-3, respectively.

[0040] 5. Dual real-time fluorescence quantitative PCR detection

[0041] Establish a dual real-time fluorescence quantitative PCR reaction system:

[0042] The reaction system is 20ul, 0.2ul each of the upstream primer and downstream primer of duck hepatitis A virus type 1, 0.4ul each of the upstream primer and downstream primer of duck astrovirus type 3, 1ul each of DNA template, and sterile deionized water is added to the final volume of 20ul. TM Amplification reaction was performed on a Real-Time System machine, and the reaction program conditions were set as follows: 95°C, 15 min pre-denaturation; 95°C 10 s, 60°C 30 s, for a total of 40 cycles; after the reaction was completed, a melting curve was generated.

[0043] 6. Establishment of standard curve

[0044] The positive standards pMD-19T-DHAV-1 and pMD-19T-DAstV-3 were serially diluted 10-fold (10 8 -10 1 The amplification was performed under the established conditions, and the standard curves of DHAV-1 and DAstV-3 were established with the copy number as the horizontal axis and the Ct value as the vertical axis.

[0045] 7. Establishment of dual fluorescence quantitative dissolution curve

[0046] Positive standards pMD-19T-DHAV-1 and pMD-19T-DAstV-3 (10 10 copies / uL) were amplified using the established dual real-time fluorescence quantitative PCR reaction system and conditions, according to the Bio-Rad CFX96 TMThe Real-Time System instrument automatically generates a melting curve.

[0047] 8. Specificity Experiment

[0048] The established reaction system and conditions were used to detect duck Tembusu virus (DTMUV), new goose parvovirus (NGPV), duck circovirus (DuCV), and Newcastle disease virus (NDV), and their specificity was evaluated.

[0049] 9. Sensitivity test

[0050] The positive standards pMD-19T-DHAV-1 and pMD-19T-DAstV-3 were serially diluted 10-fold (10 8 -10 1 ), amplified using established conditions, according to Bio-Rad CFX96 TM The Real-Time System instrument automatically generates an amplification curve and evaluates its detection limit.

[0051] Experimental results

[0052] 1. Standard curve analysis

[0053] The calibration curves for the positive standards pMD-19T-DHAV-1 and pMD-19T-DAstV-3 showed good linearity within the reaction range. The calibration curve equation for DHAV-1 was: y = -3.391x + 34.595, with a correlation coefficient of 0.997 and an amplification efficiency of 97.2%. The standard curve is shown in Figure 1. Figure 1 The standard curve equation of DAstV-3 is: y = -3.266x + 34.816, the correlation coefficient is 0.999, the amplification efficiency is 102.4%, and the standard curve is shown in Figure 2 .

[0054] 2. Melting curve analysis

[0055] From the melting curve analysis, it can be seen that DHAV-1 has a single specific peak at a Tm value of 85°C, without primer dimers and nonspecific primers (see Figure 3 ); DAstV-3 showed a single specific peak at Tm value of 80℃, without primer dimers and non-specific primers (see Figure 4 ).exist Figure 5 In the double melting curve, the Tm value of DHAV-1 was 85℃ and that of DAstV-3 was 80℃, which was consistent with the results of single melting curve analysis.

[0056] 3. Specificity experimental analysis

[0057] Under the established dual fluorescence quantitative PCR reaction system and conditions, the detection of DTMUV, NGPV, DuCV, and NDV were all negative (see Figure 6 ).

[0058] 4. Sensitivity experimental analysis

[0059] Bole CFX96 TM The Real-Time System instrument collects the fluorescence signal and automatically generates the sample amplification curve. Figure 6 As shown, at 7.34×10 8 to 7.34×10 1 The detection limit of DHAV-1 was determined to be 7.34×10 1 Copy number / μL (see Figure 1 ); at 3.78×10 8 to 3.78×10 1 The detection limit of DAstV-3 was determined to be 3.78×10 1 Copy number / μL (see Figure 2 ).

Claims

1. A SYBR-Green I fluorescent quantitative PCR primer set for detecting duck hepatitis A virus type 1 and duck astrovirus type 3, characterized in that: The primer set includes a specific primer sequence for hepatitis A virus type 1 and a specific primer sequence for duck astrovirus type 3; The specific primer sequence of the hepatitis A virus type 1 is: Upstream primer F1: AACTACACAGACAATACT; Downstream primer R1: CAGGCATAATAATCATCAT; The specific primer sequence of the duck astrovirus type 3 is: Upstream primer F2: ACGACAGTAGATAACAAC; Downstream primer R2: GTCATCACCATAACAGAT.

2. Use of the fluorescent quantitative PCR primer set according to claim 1 in preparing a reagent for detecting duck hepatitis A virus type 1 and duck astrovirus type 3.

3. A fluorescent quantitative PCR kit for detecting duck hepatitis A virus type 1 and duck astrovirus type 3, characterized in that: A reaction solution containing the primer set according to claim 1 and SYBR-Green fluorescent dye.

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