CN113355460A

CN113355460A - Primer and kit for detecting novel goose reovirus, and detection method and application thereof

Info

Publication number: CN113355460A
Application number: CN202110682628.4A
Authority: CN
Inventors: 董嘉文; 黄允真; 张俊勤; 孙敏华; 李林林; 张建峰; 廖明
Original assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Current assignee: Institute of Animal Health of Guangdong Academy of Agricultural Sciences
Priority date: 2021-06-18
Filing date: 2021-06-18
Publication date: 2021-09-07
Anticipated expiration: 2041-06-18
Also published as: CN113355460B

Abstract

The invention discloses a primer and a kit for detecting novel goose reovirus, a detection method and application thereof, wherein the primer pair and the probe for detecting the novel goose reovirus target the lambda C gene of the novel goose reovirus. The nucleotide sequences of the novel goose reovirus detection primer pair are shown as SEQ ID No.1 and 2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The Novel Goose Reovirus (NGRV) detection primer pair and the probe have strong specificity and high sensitivity, the minimum detectable limit is 56.6 copies, and the detection method has simple operation and good repeatability, can carry out quantitative analysis quickly, accurately and at high flux, and is favorable for popularization and application in clinical practice.

Description

Primer and kit for detecting novel goose reovirus, and detection method and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a primer and a kit for detecting a novel goose reovirus, and a detection method and application thereof.

Background

In recent years, with the large-scale and industrialized development of the aquatic bird breeding industry, the aquatic bird breeding density is continuously improved, but the breeding management level is relatively laggard, so that the cross infection of various pathogens among different hosts is easy to occur in the large-scale aquatic bird breeding. In addition, as part of waterfowls are common natural carriers and storage hosts of various viruses, the problems of complexity and diversification of waterfowl epidemic diseases are aggravated, and new symptoms or new infectious diseases suddenly appear in old diseases.

Since 2020, infectious diseases with punctate white focal necrosis of the liver and spleen as main pathological features appear in part of goose breeding and breeding areas, and are named as Novel Goose Reovirus (NGRV) through pathogen separation identification and sequence determination, but the whole genome sequence of the goose reovirus is not disclosed correspondingly.

In the related art, the method for detecting the aquatic bird reovirus pathogen mainly comprises the following steps: virus separation and identification, agar diffusion test, common PCR, fluorescent quantitative PCR and the like. However, at present, a rapid detection method capable of effectively aiming at the novel goose reovirus does not exist, and the epidemic situation of the virus in waterfowl groups is not yet clear, so that a reliable, effective and rapid method for detecting the novel goose reovirus in clinical samples is provided, and the method has important significance for monitoring, epidemiological investigation and prevention and control of the novel goose reovirus.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the primer, the kit and the detection method for detecting the novel goose reovirus can accurately detect the novel goose reovirus in a sample to be detected, have strong specificity and high sensitivity, and have important significance for monitoring, epidemiological investigation and prevention and control of the novel goose reovirus.

In a first aspect of the invention, Novel Goose Reovirus (NGRV) detection primers and/or probes are provided,

the novel goose reovirus detection primer is used for amplifying a specific gene segment of the novel goose reovirus;

the probe targets the lambda C gene of the novel goose reovirus.

The inventor finds that the homology of the Novel Goose Reovirus (NGRV) and other aquatic and avian reoviruses is 27.5-97.3%, and further carries out phylogenetic analysis to know that the NGRV strain and other aquatic and avian reoviruses are gathered on the same branch, wherein the genes of mu A, mu B, sigma C and sigma NS of the NGRV and the Muscovy Duck Reovirus (MDRV) ZJ2000M are positioned on the same branch; the μ NS and λ B genes are in the same branch as European GRV isolate D20/99; the genes of lambda A and sigma A are closer to the genetic distance of the Novel Duck Reovirus (NDRV); and ac is located in a relatively independent branch, but still clustered in the same large branch as the NDRV. Therefore, it can be judged that NGRV may be recombinantly produced by different waterfowl-derived reoviruses, and thus, is more difficult to identify than known waterfowl-derived reoviruses.

According to the first aspect of the present invention, in some embodiments of the present invention, the nucleotide sequences of the novel goose reovirus detection primer pair are:

an upstream primer F: 5'-CTACGCATTCTTATGAAGTC-3' (SEQ ID NO. 1);

a downstream primer R: 5'-GCAACTACCAAGAGAAAC-3' (SEQ ID NO. 2).

According to a first aspect of the invention, in some embodiments of the invention, the nucleotide sequence of the probe is:

and (3) probe P: 5'-ACTATCGCTACTGTCACCGCC-3' (SEQ ID NO. 3).

The primer pair and the probe in the invention both use the lambda C gene of the reovirus as a target gene for primer design, and the inventor compares the lambda C gene of the novel goose reovirus with other reoviruses of the same genus, selects a specific region of the novel goose reovirus as a design site of the primer, so as to ensure the specificity of the detection method. And screening the designed primers, and selecting the primer probe combination with the strongest specificity and the highest sensitivity to obtain the primer pair and the probe.

In some preferred embodiments of the invention, the probe is linked to a fluorescent reporter at the 5 'end and a fluorescent quencher at the 3' end.

In some preferred embodiments of the invention, the fluorescent reporter group comprises at least one of FAM, HEX, VIC, CY5, TET.

In some more preferred embodiments of the invention, the fluorescent reporter is FAM.

In some preferred embodiments of the invention, the fluorescence quenching group comprises at least one of BHQ, TAMRA, MGB.

In some more preferred embodiments of the invention, the fluorescence quencher group is BHQ.

Of course, those skilled in the art can also reasonably select other fluorescence reporter groups and fluorescence quencher groups according to the actual use requirements.

In a second aspect of the present invention, a novel goose reovirus detection reagent is provided, which contains the novel goose reovirus detection primer and/or probe according to the first aspect of the present invention.

The novel goose reovirus detection reagent has strong specificity and high sensitivity on the novel goose reovirus, and the minimum detection limit can reach 5.66 multiplied by 10¹The copies/mu L has important significance for monitoring, epidemiological investigation and prevention and control of the novel goose reovirus disease.

In a third aspect of the invention, the application of the novel goose reovirus detection reagent in the second aspect of the invention in the preparation of the novel goose reovirus detection kit is provided.

According to a third aspect of the present invention, in some embodiments of the present invention, the novel goose reovirus detection kit further comprises a positive control.

In some preferred embodiments of the invention, the positive control comprises a novel goose reovirus standard or positive plasmid.

In some preferred embodiments of the invention, the positive control is a p-NGRV plasmid.

According to the third aspect of the invention, in some embodiments of the invention, the use method of the novel goose reovirus detection kit comprises the following steps:

(1) extracting nucleic acid of a sample to be detected;

(2) carrying out PCR amplification by using a nucleic acid of a sample to be detected as a template and using the novel goose reovirus detection primer pair and the probe in the first aspect of the invention to obtain an amplification curve;

(3) if an amplification curve is obtained and is consistent with the positive control amplification curve, the sample to be detected contains the novel goose reovirus; otherwise, the sample to be detected does not contain the novel goose reovirus.

In some preferred embodiments of the present invention, the PCR amplification system is:

the 2 Xone Step Mix and One Step Enzyme Mix were purchased from NyVon NuoZan Biotech Inc.

Of course, the skilled person can also use the buffer solution and the premix solution in other one-step RT-PCR kits to perform amplification, or use the buffer solution and the premix solution in conventional PCR kits to perform detection after performing reverse transcription on the sample to be detected by using the conventional method in the art.

In some preferred embodiments of the invention, the PCR amplification conditions are: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 47 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle is 45 times.

The invention has the beneficial effects that:

1. the Novel Goose Reovirus (NGRV) detection primer pair and the probe have strong specificity, do not generate false negative on Goose Parvovirus (GPV), tembusu virus (TMUV), goose circovirus (GoCV), Muscovy Duck Reovirus (MDRV), Novel Duck Reovirus (NDRV) and goose astrovirus (GAstV) nucleic acid, and do not generate non-specific amplification condition.

2. The Novel Goose Reovirus (NGRV) detection primer pair and the probe have high sensitivity, are designed aiming at the gene sequence of the novel goose reovirus, and have the lowest detectable limit of 5.66 multiplied by 10¹And (4) copying.

3. The detection method provided by the invention is simple to operate, can be operated according to the traditional reverse transcription PCR, has the detection period of not more than 3 hours, is strong in specificity, high in sensitivity and good in repeatability, can be used for carrying out quantitative analysis quickly, accurately and in high flux, and is favorable for popularization and application in clinical practice.

Drawings

FIG. 1 is a graph showing the fluorescent quantitative PCR amplification curves of different viruses in the examples of the present invention, wherein no amplification curve is shown for Goose Parvovirus (GPV), Tembusu virus (TMUV), goose circovirus (GoCV), Muscovy Duck Reovirus (MDRV), novel Muscovy duck reovirus (NDRV), goose astrovirus (GAstV) and negative control, and only for p-NGRV positive plasmid;

FIG. 2 is a graph showing the amplification curves of the standard plasmid p-NGRV at different dilutions in the example of the present invention;

FIG. 3 is a standard graph of a test method in an embodiment of the invention;

fig. 4 is a graph of amplification curves of 9 clinical samples suspected of being infected with the new goose reovirus in an example of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Primer and probe design

In this example, primers and probes for detecting the novel goose reovirus were designed based on the λ C gene sequence of the novel goose reovirus.

The specific selection principle is as follows: the lambda C gene of the novel goose reovirus is used as a target gene designed by a primer, the inventor compares the lambda C gene of the novel goose reovirus with other reoviruses belonging to the same genus, and a specific region of the novel goose reovirus is selected as a design site of the primer so as to ensure the specificity of the detection method. And screening the designed primers, and selecting the primer probe combination with the strongest specificity and the highest sensitivity.

The nucleotide sequence of the primer obtained in the embodiment is as follows:

an upstream primer F: 5'-CTACGCATTCTTATGAAGTC-3' (SEQ ID NO. 1);

a downstream primer R: 5'-GCAACTACCAAGAGAAAC-3' (SEQ ID NO. 2).

The nucleotide sequence of the probe obtained in this example was:

and (3) probe P: 5'-ACTATCGCTACTGTCACCGCC-3' (SEQ ID NO. 3).

Wherein, the probe P is placed in a position with the 5 'end connected with a fluorescence reporter group and the 3' end connected with a fluorescence quenching group.

The fluorescence reporter group can be at least one of FAM, HEX, VIC, CY5 and TET, and the fluorescence quencher group can be at least one of BHQ, TAMRA and MGB.

In this example, the fluorescence reporter group used in probe P is FAM and the fluorescence quencher group is BHQ.

Positive control

And (3) culturing a virus sample which is separated and sequenced to determine as the novel goose reovirus to obtain virus liquid, extracting virus nucleic acid, and carrying out PCR amplification by using the primer F/R in the embodiment as an amplification primer.

The PCR amplification system is shown in Table 1.

TABLE 1 fluorescent quantitative PCR reaction System

Composition (I)	Content (wt.)
		2×One Step Mix	5.0μL
One Step Enzyme Mix	0.5μL
		10. mu. mol/L primer F	0.4μL
10. mu. mol/L primer R	0.4μL
		10. mu. mol/L Probe P	0.2μL
Form panel	1.0μL
		ddH₂O	2.5μL
Total amount of	10μL

Among them, One Step Mix and One Step Enzyme Mix were purchased from Nanjing Novowed Biotechnology Ltd. Of course, the premix and buffer may be replaced with other kits.

The PCR amplification reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 47 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle is 45 times.

After PCR amplification is finished, an amplification product is recovered and purified, and is cloned into a PMD18T-Vector plasmid by adopting a conventional method in the field to construct a positive standard product p-NGRV plasmid. The p-NGRV plasmid was further sequenced to confirm that the fragment was correctly inserted into the vector, the plasmid was extracted, and the concentration and purity were determined.

Test of Effect of primer Probe combination in the above examples

1. And (3) specific detection:

nucleic acids of Goose Parvovirus (GPV), tembusu virus (TMUV), goose circovirus (GoCV), Muscovy Duck Reovirus (MDRV), Novel Duck Reovirus (NDRV) and goose astrovirus (GAstV) are respectively extracted, the extracted viral nucleic acids are used as PCR templates, water is used as a negative control, the prepared p-NGRV plasmid is used as a positive control, and the primer probe combination in the embodiment is used for detection (fluorescence PCR amplification).

The PCR amplification system is shown in Table 1. The PCR amplification reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 47 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle is 45 times.

The results are shown in FIG. 1.

It was found that, in all the samples tested, only the p-NGRV plasmid was used as a template to show an amplification curve, while the nucleic acids extracted from the GPV, TMUV, GoCV, MDRV, NDRV and GAstV standards were used as templates, and no amplification curve was obtained, so that it could be demonstrated that the primer-probe combination in the above examples had excellent specificity and was capable of specifically amplifying NGRV nucleic acids.

2. And (3) sensitivity test:

(1) constructing a standard curve:

the p-NGRV plasmid (positive control) constructed in the above example was diluted 10-fold to obtain 5.66X 100 to 5.66X 10⁹The positive control samples of 10 dilutions were used for copes/. mu.L, and the positive control samples of 10 dilutions were used as the control samplesTo amplify the template, PCR amplification was performed, with 2 replicates of each dilution of positive control sample.

Based on the data for each dilution of the positive control sample, a fluorescence amplification curve was plotted, and the lowest copy number at which the recombinant plasmid could be detected was determined from the amplification curve.

The amplification curve is shown in FIG. 2.

It was found that the positive standard p-NGRV plasmid was used as a template at a dilution of 5.66X 10¹To 5.66X 10⁹Amplification curves were obtained in the range of copies/. mu.L, and it was confirmed that the detection limit of the primer-probe combination in the above examples could be up to 5.66X 10¹copies/μL。

According to the amplification curve result, a standard curve is drawn by taking the Ct value as the abscissa and the logarithm of copy number as the ordinate, and the result is shown in FIG. 3.

As shown in FIG. 3, the detection method in this embodiment is 5.66 × 10¹to-5.66X 10⁹The detection range of copies/mu L has good linear relation, and the standard curve equation is as follows:

y＝0.2864x+12.612；

wherein R is²＝0.9967。

The typical S-shaped amplification curve can be displayed by performing fluorescence PCR by taking the positive standard substance p-NGRV plasmids with different copy numbers as templates, and the typical S-shaped amplification curve is uniform in distribution and good in repeatability. By combining the amplification curve, the copy number of the lowest detectable recombinant plasmid of the detection method can be reasonably determined to be 56.6 copies, which indicates that the detection method has higher detection sensitivity.

3. The actual clinical sample detection effect is as follows:

in order to further verify the effectiveness of the detection method in the above embodiment, the present embodiment performs actual detection with an actual clinical sample as a subject.

The specific experimental steps are as follows:

nucleic Acid (RNA) was extracted from 9 visceral tissues (liver, spleen, heart, etc.) suspected to be infected with goose reovirus. The extracted nucleic acid (RNA) was used as a template, the nucleic acid of the NGRV standard (or the p-NGRV plasmid in the above example can be used) was used as a positive control, and the nucleic acid (RNA) extracted from the visceral tissue of a healthy goose was used as a negative control, and fluorescence PCR amplification was performed in the fluorescence quantitative PCR reaction system shown in Table 1. The PCR amplification reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 47 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle is 45 times.

Meanwhile, the result of each detection sample is verified by sequencing (sent to Shanghai Biotechnology engineering Co., Ltd for sequencing).

The results are shown in table 2 and fig. 4.

TABLE 29 results of detection of visceral tissue samples of geese suspected of being infected with goose reovirus

Sample numbering	Using the detection method in the above embodiment	Results of two-way sequencing	Accuracy of detection
				1	No amplification	Negative in NGRV	Correction of
2	No amplification	Negative in NGRV	Correction of
				3	No amplification	Negative in NGRV	Correction of
4	Amplification of	Positive for NGRV	Correction of
				5	Amplification of	Positive for NGRV	Correction of
6	No amplification	Negative in NGRV	Correction of
				7	No amplification	Negative in NGRV	Correction of
8	No amplification	Negative in NGRV	Correction of
				9	No amplification	Negative in NGRV	Correction of

The result shows that 2 clinical samples in 9 parts detect the positive NGRV nucleic acid, and the detection result is consistent with the sequencing result, which indicates that the application can accurately detect the positive NGRV sample and has extremely high detection accuracy.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> institute of animal health of academy of agricultural sciences of Guangdong province

<120> primer and kit for detecting novel goose reovirus, and detection method and application thereof

<130>

<160> 3

<170> PatentIn version 3.5

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Claims

1. The novel goose reovirus detection primer and/or probe is characterized in that the novel goose reovirus detection primer is used for amplifying a specific gene segment of the novel goose reovirus;

the probe targets the lambda C gene of the novel goose reovirus.

2. The novel goose reovirus detection primer and/or probe as claimed in claim 1, wherein the nucleotide sequence of the novel goose reovirus detection primer pair is as follows:

an upstream primer F: 5'-CTACGCATTCTTATGAAGTC-3' (SEQ ID NO. 1);

a downstream primer R: 5'-GCAACTACCAAGAGAAAC-3' (SEQ ID NO. 2).

3. The novel goose reovirus detection primer and/or probe as claimed in claim 1, wherein the nucleotide sequence of the probe is:

and (3) probe P: 5'-ACTATCGCTACTGTCACCGCC-3' (SEQ ID NO. 3).

4. The novel goose reovirus detection primer and/or probe as claimed in claim 3, wherein the 5 'end of the probe is connected with a fluorescence reporter group, and the 3' end of the probe is connected with a fluorescence quencher group;

the fluorescent reporter group comprises at least one of FAM, HEX, VIC, CY5 and TET;

the fluorescence quenching group comprises at least one of BHQ, TAMRA and MGB.

5. A novel goose reovirus detection reagent, which is characterized by comprising the novel goose reovirus detection primer and/or probe as claimed in any one of claims 1 to 4.

6. Use of the novel goose reovirus detection reagent of claim 5 in the preparation of a novel goose reovirus detection kit.

7. The use of claim 6, wherein the novel goose reovirus detection kit further comprises a positive control, and the positive control is preferably a novel goose reovirus standard or a positive plasmid.

8. The use of claim 6 or 7, wherein the use method of the novel goose reovirus detection kit comprises the following steps:

(1) extracting nucleic acid of a sample to be detected;

(2) carrying out PCR amplification by using a sample nucleic acid to be detected as a template and the novel goose reovirus detection primer pair and the probe of any one of claims 1-4 to obtain an amplification curve;

9. The use of claim 8, wherein the PCR amplification system is:

10. the use of claim 8, wherein the PCR amplification conditions are: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 20s, annealing at 47 ℃ for 20s, and extension at 72 ℃ for 20 s; the cycle is 45 times.