CN107299155B - Primer and probe for real-time fluorescence quantitative PCR detection of goose astrovirus - Google Patents

Abstract

The invention relates to a primer and a probe for real-time fluorescence quantitative PCR detection of goose astrovirus, belonging to the field of zooepidemiology. The invention comprises the design of specific primers and probe sequences, the construction of standard plasmids, the establishment and optimization of a real-time fluorescence quantitative PCR amplification method, the extraction of sample DNA and the detection and judgment of results. The method for detecting the primers and the probes of the real-time fluorescent quantitative PCR of the goose astrovirus has high sensitivity, good stability, strong specificity and good repeatability in the detection of the goose astrovirus, can detect 15.7 copies at least, and can be used for detecting the infection of the goose astrovirus in clinical samples.

Description

Primer and probe for real-time fluorescence quantitative PCR detection of goose astrovirus

Technical Field

The invention relates to a primer and a probe for real-time fluorescence quantitative PCR detection of goose astrovirus, belonging to the field of zooepidemiology.

Background

Astrovirus belongs to the genus astrovirus (astrovirus) of the family astrovirus (astrovidae), and is classified into two genera according to host: mammalian genus astrovirus (Mamastrovirus) and avian genus astrovirus (Avastrovirus). Avian astrovirus genus currently exists in 3 genera (Avastrovirus 1-3), including Duck astrovirus (Duck astrovirus), bird nephritis virus (Avian nephritisa virus), Turkey astrovirus (Turkey astrovirus), and the like, which represent species. Astrovirus is a single-stranded positive-stranded, membrane-free, small RNA virus whose genome is approximately 6.8kb in length and comprises, in total, from 5 'to 3': 1 non-coding region at the 5 'end of 85 nucleotides, 3 Open Reading Frames (ORFs) (ORF 1a, ORF1b, ORF 2), 1 non-coding region at the 3' end of 80 nucleotides, and 1 poly A tail of 30 nucleotides.

The duck astrovirus is one of main pathogens of duck viral hepatitis (DHV), and the DHV is an important infectious disease causing harm to the duck breeding industry, can cause ducklings to have high mortality rate, and causes great economic loss to the duck breeding industry. DHV is divided into three serotypes, namely Duck Hepatitis A Virus (DHAV), duck astrovirus type 1 (DAst V-1) and duck astrovirus type 2 (DAst V-2), and no obvious antigen cross protection exists among the three serotypes. Since the first report of DHV-II (now called DAst V-1) in the United kingdom in 1965, DAst V-1 was not reported in every country, and thus DAst V-1 was considered to be restricted to its prevalence in the United kingdom. In 2008, Fu et al reported that DAstV-1 was found in China and completed whole genome sequencing of the first DAstV-1 Chinese isolate (C-NGB). DAst V-2 was found in Liuning, 2014, et al, southern China.

In recent years, with the continuous optimization of detection technology and the expansion of detection field, the sequences of new astrovirus strains obtained from human and animals have a longer evolutionary distance with the sequences of known astrovirus strains, and more new subtypes and genotypes are possible. In 2017, the group detected that the astrovirus infection existed in the goose group (named goose astrovirus GsFJ2017 strain, GenBank accession No. MF 576430) in the disease material submitted by a certain goose farm in Fujian, the nucleotide homology of the fragment and a representative strain of avian astrovirus (turkey astrovirus type 2) was 69.1%, and the nucleotide homology of the astrovirus with a duck infectious disease (Anas penelope) was only 60.6%.

Real-time fluorescent quantitative PCR is a method of measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle in DNA amplification reaction using fluorescent chemicals. A method for quantitatively analyzing a specific DNA sequence in a sample to be detected by an internal reference method or an external reference method. The real-time fluorescence quantitative PCR is to detect the PCR process in real time through a fluorescence signal in the PCR amplification process. Since in the exponential phase of PCR amplification, there is a linear relationship between the Ct value of the template and the initial copy number of the template. The fluorescent probe method is to use a sequence-specific fluorescent labeled probe to detect a product, and the appearance of the probe method greatly improves the specificity of a quantitative PCR technology compared with the conventional PCR technology. TaqMan probes, FRET hybridization probes (fluorescence resonance energy transfer probes) and molecular Beacon are now more commonly mentioned. The TaqMan probe method is characterized in that a pair of primers is added in PCR amplification, a specific fluorescent probe is added simultaneously, the probe is specifically combined with a template, and the combination site of the probe is between the two primers. The 5 'end of the probe is marked with a fluorescence Reporter group (R), such as FAM, VIC, JOE and the like, and the 3' end is marked with a fluorescence quenching group (Quencher, Q), such as Eclipse, TAMRA and the like. When the probe is complete, the fluorescence excited by the 5 'end reporter group through the light source of the instrument is just quenched by the near-distance 3' end fluorophore group, and the instrument can not detect the fluorescence signal excited by the 5 'end reporter group (namely, the emission wavelength of the 5' fluorophore group is just the absorption wavelength of the 3 'fluorophore group, so that the energy is absorbed and transferred to the 3' fluorophore group to emit other fluorescence). Along with the PCR, when the Taq enzyme encounters a probe combined with a template in the chain extension process, the 5 ' -3 ' exonuclease activity (the activity is double-strand specificity, and a free single-strand probe is not influenced) of the Taq enzyme can cut the probe, a 5 ' end reporter group is released to be free in a reaction system, the shielding of a 3 ' end fluorescence quenching group is kept away, and a fluorescence signal emitted by the excited 5 ' end reporter group can be detected by the probe. That is, for each amplified DNA strand, a fluorescent molecule is formed, so that the accumulation of the fluorescent signal and the formation of the PCR product are completely synchronized, and the intensity of the report signal represents the copy number of the template DNA. At present, no primer, probe and method related research report for real-time fluorescent quantitative PCR detection of the goose astrovirus exists at home and abroad, and the establishment of the invention can fill the blank of related fields at home and abroad.

Disclosure of Invention

The invention aims to fill the blank of the related research reports of the existing method for detecting the primer and the probe of the goose astrovirus by the real-time fluorescent quantitative PCR, and provides the primer and the probe for the real-time fluorescent quantitative PCR detection of the goose astrovirus and the using method thereof. The method has the advantages of high sensitivity, good stability, strong specificity and good repeatability, can detect 15.7 copies at least, can be used for molecular epidemiological investigation of the goose astrovirus in clinical samples, and provides a detection method and means for determining the molecular epidemiological characteristics of the goose astrovirus.

In order to achieve the purpose, the invention adopts the following technical scheme:

an upstream primer GoAst-F: 5'-GGCCAATATTCAACAACA-3' the flow of the air in the air conditioner,

a downstream primer GoAst-R: 5'-CCTTCCTTATTGACACAAG-3', respectively;

the probe sequence GoAst-P is as follows: 5'-TGTGTAATGTCTGGCTCACCCA-3', wherein the 5 '-end is marked with a fluorescence reporter group FAM, and the 3' -end is marked with a fluorescence quenching group Eclipse.

The real-time fluorescent quantitative PCR detection method of the primers and the probes for the goose astrovirus comprises the following steps:

(1) construction and preparation of quantitative standard plasmid

The extracted goose astrovirus nucleic acid DNA is used as a template, and a gene fragment (the target fragment size is 331 bp) containing a target sequence is subjected to PCR amplification by using an upstream primer GoAst-F3 (the primer sequence is 5'-AGACACCACAGCTTAAGAAA-3') and a downstream primer GoAst-R3 (the primer sequence is 5'-ATATTTTTATACATATCTAT-3'). The primers were synthesized by Baori physician's article technology (Beijing) Co., Ltd.

Amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master MIX), in which 25. mu.L of 2 XPCR Master MIX reaction solution, upstream and downstream primers (GoAst-F3 and GoAst-R3) (primer concentration 10. mu. mol. L)^-1) mu.L of each 1. mu.L of the extracted nucleic acid template DNA was supplemented with sterile deionized water to a final reaction system of 50. mu.L. Mixing, performing PCR amplification under the conditions of pre-denaturation at 95 deg.C for 5 min, circulating, denaturation at 94 deg.C for 50 s, annealing at 51 deg.C for 30s, extension at 72 deg.C for 35s, and final extension at 72 deg.C for 10 min after 35 cycles.

After the reaction is finished, according to the conventional agarose gel electrophoresis identification, the Universal DNA purification recovery kit is used for tapping and recovering the target band which is expected by experimental amplification, and a cloning vector (pEASY-T1 Simple cloning kit) is used for cloning the gel recovery fragment. Transforming DH5a clone competent cells, coating a plate overnight, randomly picking 12 single colonies, shaking the colonies for 14h, extracting corresponding plasmids, carrying out double enzyme digestion identification, sending the plasmids to Shanghai Boshang biotechnology Limited company for sequence determination, determining the sequence determination result as a goose astrovirus sequence through BLAST analysis, and taking the positive recombinant plasmid as a standard plasmid (T-GoAst).

(2) Real-time fluorescent quantitative PCR reaction system and procedure:

using goose astrovirus positive standard (T-GoAst) as a template, and carrying out serial dilution (the plasmid concentration is 1.57 multiplied by 10)⁶、1.57×10⁵、1.57×10⁴、1.57×10³、1.57×10²And 1.57X 10¹Copies/. mu.L), real-time fluorescent quantitative PCR reactions were performed at different annealing temperatures (54, 56, 58, 60, 62 and 64 ℃), primer (GoAst-F, GoAst-R) concentrations (2.5, 5.0, 10 and 20. mu. mol/L) and probe (GoAst-P) concentrations (1.25, 2.5, 5 and 10. mu. mol/L), and the reaction conditions were optimized. And (5) judging the result, namely observing amplification of a positive fluorescence signal related to the FAM signal, and judging that the sample to be detected is positive for the goose astrovirus infection if the positive fluorescence signal exists.

The optimal 25 muL reaction system optimized by the established detection method of the primers and the probes for real-time fluorescence quantitative PCR detection of the goose astrovirus is as follows: premix Ex Taq (Probe qPCR) mixture 12.5. mu.L, upstream/downstream primers (GoAst-F, GoAst-R) (10. mu. mol/L) each 0.5. mu.L, Probe (GoAst-P) (5. mu. mol/L) 1. mu.L, template 2. mu.L, and water to 25. mu.L. The optimized double real-time fluorescent quantitative PCR method has the following optimal reaction conditions: pre-denaturation at 95 ℃ for 60 s; annealing at 95 ℃ for 10s, annealing at 58 ℃ for 10s, and extending at 72 ℃ for 15 s for 40 cycles.

And (5) carrying out amplification by using the optimized reaction conditions to obtain an amplification kinetic curve. And (3) deducing a standard linear regression equation (standard curve) by taking the common logarithm (lgC) of the initial copy number of the standard substance as an abscissa and taking a cycle threshold (Ct value) as an ordinate, and obtaining sensitivity test data of the standard substance.

As can be seen from FIG. 1, the lowest detection limit of the established real-time fluorescent quantitative PCR method is 15.7 copies/. mu.L. Taking the common logarithm (lgC) of the plasmid content (C) in each standard product as an abscissa and taking a cycle threshold (Ct value) as an ordinate, obtaining a goose astrovirus real-time fluorescence quantitative PCR standard curve (shown in figure 2), wherein the slope of the obtained standard curve is-3.241, the Y-axis intercept is 36.49, the correlation coefficient is 0.998, the amplification efficiency is 99.9%, and the standard curve accords with experimental expectation.

Advantageous effects

The invention adopts the primer and the probe for real-time fluorescent quantitative PCR detection of the goose astrovirus to detect the goose astrovirus, and has the following advantages and effects:

1. the detection is rapid and efficient: the detection method does not need to carry out conventional agarose gel electrophoresis detection, and the result can be judged by a program carried by a real-time fluorescent quantitative PCR machine after the reaction is finished. Only 2h is needed from nucleic acid extraction to result determination, and 96 sample detections can be simultaneously performed at one time. After detecting 75 clinically collected duck pathogens, 4 goose astrovirus infections are detected.

2. The quantification is accurate: by preparing a standard substance and drawing a standard curve, the infected goose astrovirus is directly and accurately quantified according to the Ct value of the goose astrovirus in a sample to be detected.

3. The sensitivity is high: the minimum 15.7 copies can be detected, and the detection sensitivity is improved by 100 times compared with the conventional PCR detection.

4. The specificity is strong: and common infectious diseases in goose groups (such as goose colibacillosis, goose parvovirus disease, goose reovirus disease and goose polyoma virus disease) have no response signals, and only the goose astrovirus has a fluorescence signal when detected.

5. The repeatability is good: the intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method for detecting the goose astrovirus is 0.43-1.89%, and the inter-group variation coefficient is 0.49-2.31%, which shows that the established real-time fluorescent quantitative PCR detection method has good repeatability.

Drawings

FIG. 1 is an amplification curve of a PCR method for real-time fluorescence quantitative detection of goose astrovirus. 1: 1.57X 10⁶Copy/. mu.L; 2: 1.57X 10⁵Copy/. mu.L; 3: 1.57X 10⁴Copy/. mu.L; 4: 1.57X 10³Copy/. mu.L; 5: 1.57X 10²Copy/. mu.L; 6: 1.57X 10¹Copies/. mu.L.

FIG. 2 is a standard curve of PCR method for real-time fluorescence quantitative detection of goose astrovirus.

FIG. 3 shows the specificity of the PCR method for real-time fluorescence quantitative detection of goose astrovirus. 1: goose astrovirus; 2: goose escherichia coli; 3: goose parvovirus; 4: goose reovirus; 5: goose polyoma virus.

Detailed Description

The following examples further illustrate the invention.

Example 1

1. Design and Synthesis of primers and probes

Specific primers (GoAst-F and GoAst-R) and a probe (GoAst-P) of a PCR method for real-time fluorescent quantitative detection of the goose astrovirus are designed by referring to the gene sequence characteristics of polymerase 1b protein (polymerase 1b protein) of the goose astrovirus (the goose astrovirus GsFJ2017 strain, GenBank accession number is MF 576430), wherein the primer sequences of the GoAst-F and GoAst-R are as follows:

an upstream primer GoAst-F: 5'-GGCCAATATTCAACAACA-3' the flow of the air in the air conditioner,

a downstream primer GoAst-R: 5'-CCTTCCTTATTGACACAAG-3', respectively;

the probe sequence GoAst-P is as follows: 5'-TGTGTAATGTCTGGCTCACCCA-3', wherein the 5 '-end is marked with a fluorescence reporter group FAM, and the 3' -end is marked with a fluorescence quenching group Eclipse. Primers and probes were synthesized by Baori physician technology (Beijing) Inc.

1. Construction of standards

According to the characteristics of the gene sequence coded by polymerase 1b protein of the goose astrovirus (goose astrovirus GsFJ2017 strain) identified in the early stage of the team, a specific primer is designed by utilizing primer design software Oligo (version v7.37), wherein the primer sequence is as follows: GoAst-F3: 5'-AGACACCACAGCTTAAGAAA-3' and GoAst-R3: 5'-ATATTTTTATACATATCTAT-3', used for amplifying polymerase 1b protein gene fragment of about 331bp, and the primers were synthesized by Baozi physician's technology (Beijing) Co., Ltd.

Goose astrovirus (GsFJ 2017 strain) nucleic acid RNA was extracted using the Viral nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit, and RT-PCR was performed according to the 50. mu.L system recommended by PrimeScript [ One Step RT-PCR Kit Ver.2 (Dye Plus), wherein 2. mu.L of PrimeScript 1 Step Enzyme Mix reaction solution, 25. mu.L of 2X 1 Step Buffer (Dye Plus) reaction solution, 2. mu.L of upstream and downstream primers (GoAst-F3 and GoAst-R3, 10. mu.M), 2. mu.L of the extracted nucleic acid RNA, and a final volume of 50. mu.L of deionized water were supplemented. The reaction conditions are as follows: carrying out reverse transcription at 50 ℃ for 30 min and then carrying out a PCR amplification program; pre-denaturation at 94 ℃ for 4 min; at 94 ℃ for 50 s, at 56 ℃ for 30s, at 72 ℃ for 45 s, for 35 cycles; after the circulation is finished, the extension is carried out for 10 min at 72 ℃.

And identifying the PCR product by using 1.0% agarose gel electrophoresis, and cutting and recovering the specific target fragment by using an agarose gel recovery kit. The RT-PCR amplified specific polymerase 1b protein gene fragment was cloned on pEASY-T1 Cloning vector according to pEASY-T1 Simple Cloning Kit instructions, 8 single colonies were randomly picked up and cultured in ampicillin (content 100. mu.g/mL) resistant LB liquid medium for 14h, and then the corresponding plasmid was extracted using fast plasmid mini-extraction Kit. The extracted plasmids are subjected to PCR identification by using primers (GoAst-F3 and GoAst-R3) and conditions during RT-PCR amplification, and the screened positive recombinant plasmids are sent to the doctor of Baozi technology (Beijing) Limited for sequencing. And carrying out BLAST analysis verification on the sequencing result on NCBI, taking the positive recombinant plasmid which is in line with the test expectation as a positive standard (T-GoAst) of the real-time fluorescence quantitative PCR, subpackaging and storing at-20 ℃ for later use.

3. Real-time fluorescent quantitative PCR reaction condition

The optimal 25 muL reaction system optimized by the established detection method of the primers and the probes for real-time fluorescence quantitative PCR detection of the goose astrovirus is as follows: premix Ex Taq (Probe qPCR) mixture 12.5. mu.L, upstream/downstream primers (GoAst-F and GoAst-R) (10. mu. mol/L) each 0.5. mu.L, Probe (GoAst-P) (5. mu. mol/L) 1. mu.L, template 2. mu.L, and water to 25. mu.L. The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; 10s at 95 ℃, 10s at 56 ℃ and 15 s at 72 ℃ for 40 cycles.

4. Specificity detection

And common infectious diseases in goose groups (such as goose escherichia coli, goose parvovirus, goose reovirus and goose polyoma virus) have no reaction signals, and only a fluorescence signal appears when the goose astrovirus is detected.

5. Repeatability test

The intra-group variation coefficient of the real-time fluorescence quantitative PCR method for detecting the standard positive sample is 0.43-1.89%, the inter-group variation coefficient is 0.49-2.31%, and the result is shown in Table 1, which indicates that the established real-time fluorescence quantitative PCR detection method has good repeatability.

TABLE 1 real-time fluorescent quantitative PCR coefficient of variation

6. Clinical application

After detecting 75 clinically collected duck pathogens by using the established detection method of the primers and the probes for real-time fluorescent quantitative PCR detection of the goose astrovirus, 4 goose astrovirus infections are detected to be positive, and the positive rate is 5.33%.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims (2)

1. A primer and a probe for real-time fluorescence quantitative PCR detection of goose astrovirus are characterized in that: the primer sequences are as follows:

an upstream primer GoAst-F: 5'-GGCCAATATTCAACAACA-3' the flow of the air in the air conditioner,

a downstream primer GoAst-R: 5'-CCTTCCTTATTGACACAAG-3', respectively;

the probe sequence GoAst-P is as follows: 5'-TGTGTAATGTCTGGCTCACCCA-3', wherein the 5 '-end is marked with a fluorescence reporter group FAM, and the 3' -end is marked with a fluorescence quenching group Eclipse.

2. A real-time fluorescence quantitative PCR detection kit for goose astrovirus is characterized in that: the kit comprises the primer and the probe of claim 1.

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