Multiplex fluorescent PCR detection primer probe set, kit and application for different genotypes of goose astrovirus

Abstract

The application discloses a different genotype goose astrovirus multiplex fluorescent PCR detection primer probe set, a kit and application, relates to the field of molecular biology; the primer probe set comprises a primer pair and a probe for detecting type 1 goose astrovirus (GoAstV-1) and type 2 goose astrovirus (GoAstV-2); the primer pair for detecting GoAstV-1 is as shown in SEQ ID NO. 1-2, and the probe is as shown in SEQ ID NO. 3; the primer pair for detecting GoAstV-2 is as shown in SEQ ID NO. 4-5, and the probe is as shown in SEQ ID NO. 6. The primer probe set disclosed by the application can realize multiplex fluorescent PCR detection of different genotypes of goose astrovirus, the detection method has good specificity, sensitivity and repeatability, and provides technical support for clinical diagnosis of GoAstV, determination of virus load and epidemiological investigation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to a goose astrovirus multiplex fluorescent PCR detection primer probe set, a kit and an application thereof. BACKGROUND

[0002] Goose astrovirus disease is a fatal infectious disease caused by goose astrovirus (GoAstV) infection, which is mainly characterized by multi-species goose joint and visceral gout. The highest infection rate and mortality rate of the diseased goose group can reach 80% and 50% respectively, causing huge economic losses to the goose industry. GoAstV mainly infects goslings under 3 weeks old, causing a large amount of urate deposition on the surface of heart, liver, kidney and joint cavity. In addition, GoAstV can also infect other poultry such as chickens and ducks, indicating that GoAstV has the possibility of cross-species transmission, which brings new challenges to the prevention and control of the disease.

[0003] GoAstV belongs to the Astroviridae family and is a non-enveloped single-stranded RNA virus. According to the different hosts infected by astrovirus, it is divided into two genera, Mamastrovirus (MAstV) and Avastrovirus (AAstV). At present, the International Committee on Taxonomy of Viruses (ICTV) ninth report divides the avian astrovirus genus into three groups, namely avian astrovirus group 1 (AAstV-1), avian astrovirus group 2 (AAstV-2) and avian astrovirus group 3 (AAstV-3). GoAstV belongs to avian astrovirus group 1 and is a newly emerging astrovirus. The first full-length genome sequence of GoAstV was reported by Zhang Dapang in 2017, named FLX. Since then, multiple full-length genome sequences of GoAstV have been reported, such as GD, SDPY, HN1G, SD01, GsFJ02, etc. Genetic evolution analysis shows that all known GoAstVs sequences belong to two different phylogenetic branches. According to the genetic evolution relationship, GoAstV is divided into two types, FLX-like strains are named GoAstV-1, and SD01-like strains are named GoAstV-2, also known as new GoAstV. Due to the lack of suitable cell culture system for GoAstV-1 strain proliferation, the GoAstV-1 strain is difficult to isolate and culture, and its pathogenicity is also difficult to verify. The first goose astrovirus strain (SD-01) was isolated by Zhang Qingshui et al. in 2018, and then multiple GoAstV-2 strains were successfully isolated, such as SDPY, GD, CXZ18, etc.

[0004] The main laboratory diagnostic methods of GoAstV are virus isolation and identification, electron microscopy and molecular biology methods. Virus isolation and identification is the traditional detection method of GoAstV, which mainly involves inoculation of goose embryos or chicken hepatoma cells. Generally, it needs to be transmitted to 3 generations or so before the goose embryo dies or obvious lesions appear, which is time-consuming. Electron microscopy is one of the early detection methods of astrovirus. However, Yuan et al. used goose liver ultrathin sections for observation, but could not observe the specific morphology of astrovirus, so this method has certain defects. The molecular biology detection methods of GoAstV mainly include RT-PCR, fluorescent quantitative RT-PCR and RT-LAMP, etc. RT-PCR method can directly detect GoAstV in goose diseased tissues, and has high sensitivity and good specificity. The diagnosis of GoAstV-1 mainly focuses on the preparation of polyclonal antibody. Liu et al. used prokaryotic expression system to express GoAstV-1 structural protein ORF2 and prepared its polyclonal antibody. However, there are few reports on the molecular biology diagnosis technology of GoAstV-1. At present, the diagnosis of GoAstV mainly focuses on GoAstV-2. Yu Mingxing et al. designed and screened specific amplification primers according to the ORF2 gene sequence, optimized the amplification conditions, and established the RT-PCR method for detecting GoAstV-2. This method has strong specificity and the detection sensitivity is 62 fg / μL. Zhang Yuxia et al. designed a set of specific LAMP primers according to the conserved sequence of GoAstV ORF1b gene, and established the LAMP rapid detection method of GoAstV-2. This method has strong specificity and the sensitivity is 1 ng / μL. RT-PCR and RT-LAMP cannot quantitatively detect them, so establishing a rapid, sensitive and specific fluorescent quantitative molecular diagnosis method is one of the main measures to effectively prevent and control the disease. At present, some researchers have established SYBR GreenⅠreal-time fluorescent quantitative PCR method. Yuan et al. established GoAstV-2 real-time fluorescent quantitative PCR method, but compared with this method, TaqMan probe fluorescent quantification has obvious advantages in absolute quantification of target genes. Su Shibobo et al. established GoAstV-2 TaqMan fluorescent quantitative PCR detection method, which has strong specificity and the detection limit is 1.5×10 2 copies / μL.

[0005] Currently, there are no treatments or vaccines for this disease, and the focus of prevention and control is on the detection and control of the epidemic. Therefore, it is particularly important to establish a rapid, accurate and time-saving detection method for epidemic prevention and control. TaqMan real-time quantitative PCR has the advantages of high specificity and high sensitivity, and has obvious advantages in the absolute quantification of target genes. It has been widely used in the clinical diagnosis of animal diseases and the determination of viral load. According to the results of epidemiological surveys, mixed infection of type 1 goose astrovirus and type 2 goose astrovirus is serious. Therefore, this invention aims to establish a real-time real-time quantitative PCR method for different genotypes of GoAstV using TaqMan, to provide technical support for the clinical diagnosis of GoAstV, the determination of viral load and epidemiological surveys. Multiplex fluorescent PCR can simultaneously perform qualitative and quantitative detection of two or more pathogens in one reaction, but it is difficult to implement, mainly in the following aspects: (1) It is necessary to design conserved primers and probes for different viruses; (2) The primers of multiplex PCR should not interfere with each other and the probes should not interfere with each other; (3) The technical parameters such as qPCR amplification efficiency and detection sensitivity should not be greatly affected by multiplex PCR. Summary of the Invention

[0006] The purpose of this invention is to provide primer and probe sets, kits, and applications for multiplex fluorescent PCR detection of different genotypes of goose astrovirus, in order to solve the problems existing in the prior art. Using the primer and probe set of this invention, multiplex fluorescent PCR detection of different genotypes of goose astrovirus can be achieved, and the detection method has good specificity, sensitivity, and repeatability.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a primer and probe set for multiplex fluorescent PCR detection of different genotypes of goose astrovirus, including primer pairs and probes for detecting GoAstV-1 and GoAstV-2; the primer pairs for detecting GoAstV-1 are shown in SEQ ID NO.1-2, and the probe is shown in SEQ ID NO.3; the primer pairs for detecting GoAstV-2 are shown in SEQ ID NO.4-5, and the probe is shown in SEQ ID NO.6.

[0009] The present invention also provides the application of the above-mentioned primer and probe set in the preparation of multiplex fluorescent PCR detection kits for different genotypes of goose astrovirus.

[0010] The present invention also provides a multiplex fluorescent PCR detection kit for different genotypes of goose astrovirus, comprising the above-mentioned primer and probe set.

[0011] Further, the kit further comprises standard plasmids of GoAstV-1 and GoAstV-2, wherein the standard plasmid of GoAstV-1 contains a sequence as shown in SEQ ID NO. 7; and the standard plasmid of GoAstV-2 contains a sequence as shown in SEQ ID NO. 8.

[0012] Further, the reaction system of multiplex fluorescent PCR of the kit is as follows: 10 pmol / μL GoAstV-1 upstream primer 0.4 μL, 10 pmol / μL GoAstV-1 downstream primer 0.4 μL, 10 pmol / μL GoAstV-1 probe 0.2 μL, 10 pmol / μL GoAstV-2 upstream primer 0.4 μL, 10 pmol / μL GoAstV-2 downstream primer 0.4 μL, 10 pmol / μL GoAstV-2 probe 0.2 μL, Template 2 μL, 2×AceQ qPCR Probe Master Mix 10 μL, 50×ROXReference Dye 20.4 μL, 50 mmol / L MgCl2 1 μL, and ddH2O is supplemented to 20 μL.

[0013] Further, the reaction procedure of multiplex fluorescent PCR of the kit is as follows: 95℃ for 5 min; 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles.

[0014] The present application discloses the following technical effects:

[0015] The present application provides a primer probe set for multiplex fluorescent PCR detection of different genotypes of goose astrovirus, and the primer probe set can realize real-time fluorescent quantitative PCR detection of different genotypes of GoAstV TaqMan, the detection method has good specificity, sensitivity and repeatability, and provides technical support for clinical diagnosis, virus load determination and epidemiological investigation of GoAstV. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0017] Figure 1 GoAstV-1 amplification curve (primers and probes are shown in SEQ ID NO. 1-3); wherein, 1-9 represent 10 1 -10 9 DNA copy number / μL standard plasmid;

[0018] Figure 2 Standard curve of GoAstV-1;

[0019] Figure 3 Amplification curve of GoAstV-2 (primers and probes are shown as SEQ ID NO. 4-6); wherein, 1-9 represent 10 1 -10 9 DNA copy number / μL standard plasmid;

[0020] Figure 4 Standard curve of GoAstV-2;

[0021] Figure 5 Combined standard curve of GoAstV-1 and GoAstV-2;

[0022] Figure 6 Amplification curve obtained by using primers and probes shown as SEQ ID NO. 9-14, wherein, A is GoAstV-1; B is GoAstV-2;

[0023] Figure 7 Specificity test of multiplex fluorescent PCR; wherein, 1: GoAstV-1; 2: GoAstV-2; 3: goose parvovirus (GPV); 4: goose paramyxovirus (NDV); 5: avian reovirus (ARV); 6: negative control;

[0024] Figure 8 Sensitivity test of multiplex fluorescent PCR; wherein, 1-9 represent 10 8 -10 0 DNA copy number / μL standard plasmid; 10 represents negative control; A is GoAstV-1; B is GoAstV-2;

[0025] Figure 9 Reproducibility test results; wherein, A is GoAstV-1; B is GoAstV-2;

[0026] Figure 10 Detection of GoAstV-1 and GoAstV-2 in clinical samples;

[0027] Figure 11 Detection of 2-type GoAstV cytotoxicity CT value. DETAILED DESCRIPTION

[0028] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is not to be considered limiting in scope, but rather as being illustrative of certain aspects of the present application, and is understood that various modifications can be made by those skilled in the art.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentration, intensity, and time, an intermediate value of the range is specifically contemplated. Each of these intermediate values is also specifically contemplated. The upper and lower limits of these smaller ranges can independently be included or excluded in the ranges.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art.

[0031] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples in the specification are exemplary only and are not intended to be limiting.

[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0033] Example 1

[0034] 1. Primer design method

[0035] By searching the NCBI nucleic acid database, a total of 7 sequences of the full genome of goose astrovirus type 1 (GoAstV-1) were found. The full genome sequences were downloaded and subjected to multiple sequence alignment using mafft software, and primers were designed according to the regions with higher homology. The Accession No. of the 7 sequences are as follows:

[0036] OL762471.1, OL762472.1, OK571391.1, MH410610.1, MW340534.1, MW353015.1, KY271027.1.

[0037] The same method, NCBI nucleic acid database search, a total of 62 type 2 goose astrovirus (GoAstV-2) full genome sequence was found, download the whole genome sequence and use mafft software for multiple sequence alignment, and then design primers according to the higher homology region. The accession numbers of the 62 sequences are as follows:

[0038] MZ576222.1, MW592379.1, OK571390.1, MW413813.1, MW592377.1, MW592378.1, MZ367612.1, OK571389.1, MN307117.1, MN307119.1, MT708902.1, MN307120.1, MN428645.1, MW345727.1, MN428642.1, MN127956.1, MN127957.1, MN127958.1, MN127954.1, MN127955.1, MN127952.1, MN127953.1, OM273305.1, OM273306.1, OM273304.1, OM273302.1, OM273303.1, MZ540211.1, MN175321.1, MN307116.1, MN428643.1, MT934439.1, MT934438.1, MT934437.1, OM273308.1, OM273309.1, OM273307.1, OM273310.1, MN307118.1, MN428644.1, MN428641.1, MN307114.1, MN307115.1, MN894548.1, MK125058.1, MG934571.1, MN109955.1, MN109956.1, MN103532.1, MN109957.1, MN127951.1, MN109954.1, MF772821.1, MN068024.1, MN068023.1, MH052598.1, MH807626.1, MN399857.1, MN809622.1, MN337323.1, MN127959.1, KY807085.1.

[0039] The sequences of the designed primers and probes are shown in Table 1, and the multiplex RT-qPCR reaction system is shown in Table 2.

[0040] Table 1 Primer and probe sequences

[0041]

[0042]

[0043] Table 2 Multiplex RT-qPCR reaction system

[0044]

[0045] Total system: 20 μL, a. Add 1 μL of each template, b. Dye 2 for ABI 7500 instrument.

[0046] 2. Fluorescent quantitative PCR reaction conditions (see Table 3)

[0047] Table 3 Fluorescent quantitative PCR reaction conditions

[0048]

[0049]

[0050] 3. Standard plasmid preparation

[0051] According to the amplification region of the primer in Table 1, and appropriately extending the upstream and downstream sequences, the target sequence was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for full gene synthesis and plasmid construction.

[0052] The sequence of the full-length fragment synthesized for type 1 is as follows, wherein the bold part is the upstream and downstream primer binding sequence, and the underlined part is the probe binding sequence:

[0053]

[0054] The sequence of the full-length fragment synthesized for type 2 is as follows, wherein the bold part is the upstream and downstream primer binding sequence, and the underlined part is the probe binding sequence:

[0055]

[0056] After the synthesis of the plasmid, the concentration of the plasmid was determined to calculate the copy number of the plasmid, and then 10 9 ~ 10 1 DNA copies / μL.

[0057] 4. Multiplex fluorescent PCR

[0058] (1) Establishment of multiplex fluorescent PCR method

[0059] According to the system in Table 2, various components were added, and fluorescent quantitative PCR was performed, and the reaction program is shown in Table 3. The template was 10 1 ~ 10 9Copies / μL standard plasmid. According to the results, a standard curve was plotted, and a standard equation was obtained. Among them, the primers and probes shown in SEQ ID NO. 1-SEQ ID NO. 6 can well realize multiplex PCR amplification, the amplification curve of type 1 is shown in Figure 1 , the standard curve of type 1 is shown in Figure 2 ; the amplification curve of type 2 is shown in Figure 3 , the standard curve of type 2 is shown in Figure 4 , and the standard curve of type 1 and type 2 is shown in Figure 5 .

[0060] The standard equation of GoAstV-1 is Y=-3.54X+40.568, R 2 =0.999, Eff=91.629.

[0061] The standard equation of GoAstV-2 is Y=-3.36X+39.471, R 2 =0.998, Eff=98.439.

[0062] SEQ ID NO. 9-14, SEQ ID NO. 15-20 and SEQ ID NO. 21-26 are failed primers and probes, and the amplification results using SEQ ID NO. 9-14 are shown in Figure 6 .

[0063] (2) Specificity test

[0064] Using the multiplex fluorescent quantitative PCR method established in step (1), the nucleic acids of GoAstV-1, GoAstV-2, GPV, NDV and ARV were subjected to fluorescent quantitative PCR amplification, and a negative control without template was set up to test the specificity of the method.

[0065] The specificity test results are shown in Figure 7 , and the results show that a total of 5 templates and 1 negative control were tested, and only GoAstV-1 and GoAstV-2 were amplified, and the others were not amplified, indicating that the method has good specificity.

[0066] (3) Sensitivity test

[0067] Select 9 dilution (10 0 -10 8 ) standards as templates and water as a negative control without template, and perform fluorescent quantitative PCR amplification to determine the minimum detection amount of GoAstV-1 and GoAstV-2, and at the same time, use conventional PCR for detection to compare the sensitivity.

[0068] The sensitivity test results are shown in Figure 8 , 101 ~10 8 Standard products all have amplification curves. While 10 0 copies of the template, slightly raised after 38 cycles, indicating that the minimum detection of this method can reach 10 1 copies / μL.

[0069] (4) Reproducibility test

[0070] Select 5 dilutions (10 4 ~10 8 ) standard products in (1) for fluorescence quantitative PCR reaction, repeat amplification 3 times, and each dilution is tested 3 times under the same amplification conditions, calculate the coefficient of variation, and evaluate the repeatability of batch detection. Three positive samples from different regions were constructed into recombinant plasmids pMD-GoAstV-1, pMD-GoAstV-2, respectively, and then diluted for fluorescence quantitative PCR detection, and the coefficient of variation was calculated to evaluate the repeatability of batch detection.

[0071] The repeatability test results are shown in Table 4 and Figure 9 , the coefficient of variation of GoAstV-1 is 0.123-1.015%; the coefficient of variation of GoAstV-2 is 0.131-0.913%; indicating that the repeatability of this method is good.

[0072] Table 4 Reproducibility test of multiplex fluorescence PCR

[0073]

[0074]

[0075] Example 2 Detection of clinical samples

[0076] 70 samples of goose gout from goose farms in Jiangxi area from 2020 to 2022 were detected by multiplex fluorescence quantitative PCR, and the detection results of GoAstV-1 and GoAstV-2 in clinical samples are shown in Figure 10 , and the cell toxicity CT value detection results of GoAstV-2 are shown in Figure 11 .

[0077] The above examples only describe the preferred mode of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A primer and probe set for multiplex fluorescent PCR detection of different genotypes of goose astrovirus, characterized in that, It includes primer pairs and probes for detecting GoAstV-1 and GoAstV-2; the primer pairs for detecting GoAstV-1 are shown in SEQ ID NO.1-2, and the probe is shown in SEQ ID NO.3; the primer pairs for detecting GoAstV-2 are shown in SEQ ID NO.4-5, and the probe is shown in SEQ ID NO.

6.

2. The application of the primer and probe set as described in claim 1 in the preparation of multiplex fluorescent PCR detection kits for different genotypes of goose astrovirus.

3. A multiplex fluorescent PCR detection kit for different genotypes of goose astrovirus, characterized in that, Includes the primer-probe set as described in claim 1.

4. The multiplex fluorescent PCR detection kit for different genotype goose astroviruses according to claim 3, characterized in that, The kit also includes standard plasmids for GoAstV-1 and GoAstV-2, wherein the standard plasmid for GoAstV-1 contains the sequence shown in SEQ ID NO.7; and the standard plasmid for GoAstV-2 contains the sequence shown in SEQ ID NO.

8.

5. The multiplex fluorescent PCR detection kit for different genotype goose astroviruses according to claim 3, characterized in that, The reaction system for multiplex fluorescent PCR in the kit is as follows: 0.4 μL of 10 pmol / μL GoAstV-1 upstream primer, 0.4 μL of 10 pmol / μL GoAstV-1 downstream primer, 0.2 μL of 10 pmol / μL GoAstV-1 probe, 0.4 μL of 10 pmol / μL GoAstV-2 upstream primer, 0.4 μL of 10 pmol / μL GoAstV-2 downstream primer, 0.2 μL of 10 pmol / μL GoAstV-2 probe, 2 μL of template, 10 μL of 2×AceQ qPCR Probe Master Mix, 20.4 μL of 50×ROX Reference Dye, 1 μL of 50 mmol / L MgCl2, and ddH2O to a final volume of 20 μL.

6. The multiplex fluorescent PCR detection kit for different genotype goose astroviruses according to claim 3, characterized in that, The reaction program for multiplex fluorescent PCR in the kit is as follows: 95℃ for 5 min; 95℃ for 10 sec, 60℃ for 30 sec, for 40 cycles.

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