CN111826471A - N-DHCLV real-time fluorescent quantitative PCR detection primer and kit - Google Patents
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Abstract
The invention relates to a primer and a kit for detecting N-DHCLV (dehydroepiandrosterone-binding-polymerase chain reaction) real-time fluorescent quantitative PCR (polymerase chain reaction), belonging to the field of zooepidemics. The invention comprises the design of specific primers and probe sequences, the construction of standard plasmids, the establishment and optimization of a real-time fluorescent quantitative PCR amplification method and the detection and judgment of results. The real-time fluorescent quantitative PCR method for detecting N-DHCLV, which is established by using the primers and the probe, has the advantages of high sensitivity, good stability, strong specificity and good repeatability in the detection of N-DHCLV, can detect 55.93 copies at least, and can be used for detecting the infection of the N-DHCLV in a clinical sample.
Description
Technical Field
The invention relates to a primer and a kit for detecting N-DHCLV (dehydroepiandrosterone-binding-polymerase chain reaction) real-time fluorescent quantitative PCR (polymerase chain reaction), belonging to the field of zooepidemics.
Background
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 beacons (molecular Beacon) are currently 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, BHQ1 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.
Flaviviridae family (Flaviviridae Family) Is a family of small enveloped viruses with single positive stranded RNA genomes, most of which infect mammals and birds, and many of which are host-specific and pathogenic. Phylogenetic relationships based on the amino acid sequence of the conserved domain of RdRP indicate that members of the Flaviviridae group are clustered in 4 currently designated genera of viruses: (Flaviviridae genusFlavivirus genus) Pestiviruses (b) and (b)Pestivirus genus) Hepatitis C virus genus (A), (B)Hepacivirus genus) Pergivirus genus (A), (B), (CPegivirus genus). Wherein, Tamanna bat virus (b)Tamana bat virus) Assigned to a separate branch, was tentatively listed as a potential member of the flaviviruses (flaviviruses). In recent years, with the widespread use of high-throughput sequencing and serological methods, many researchers have found a variety of novel HCV homologous viruses (HCV-like viruses) in different mammals. A novel duck-derived hepatitis C-like virus (N-DHCLV) (Chu L, et al. A highlyvariant hepatitis-like virus in domesticducks. J Gen virol.2019, 100(8):1234-1240. doi: 10.1099/jgv.0.001298) is a newly discovered HCV-like virus (HCV-like virus) that has been discovered in recent years from the egg-laying genetic duck population. The genomics research shows that the genome is single-strand positive-strand RNA, the length of the genome is 11422bp, an Open Reading Frame (ORF) with the length of 10824bp is coded, and the length of the coded polyprotein is 3607 amino acids. Genetic evolutionary analysis shows that the virus belongs to the members of the hepatitis C virus-like genus.
At present, no primer, probe and method related research report for real-time fluorescent quantitative PCR detection of N-DHCLV is found 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 report of the existing method for detecting the primer and the probe of the N-DHCLV by the real-time fluorescent quantitative PCR, and provides the primer and the probe for the real-time fluorescent quantitative PCR detection of the N-DHCLV and the using method thereof. The method has the advantages of high sensitivity, good stability, strong specificity and good repeatability, can detect 39.04 copies at least, can be used for molecular epidemiological investigation of N-DHCLV in clinical samples, and provides a detection method and means for determining the molecular epidemiological characteristics of the N-DHCLV.
In order to realize the purpose, the following technical scheme is adopted:
a primer and a probe for real-time fluorescent quantitative PCR detection of N-DHCLV are disclosed, wherein the primer sequence is as follows:
the upstream primer N-DHCLV-T-F: 5'-TGACCCAAACACCAACTTCG-3' the flow of the air in the air conditioner,
the downstream primer N-DHCLV-T-R: 5'-TTCAGTCGCTTCCAATCCAG-3', respectively;
the probe N-DHCLV-T-P is as follows: 5'-AGTCAGAAAATTGTCCCGAGCAGGC-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.
An N-DHCLV real-time fluorescence quantitative PCR detection kit comprises the primer and the probe.
The established real-time fluorescence quantitative PCR detection method of the N-DHCLV has a reaction system of 20 mul: premix Ex Taq (Probe qPCR) mixture 10. mu.L, each of upstream and downstream primers (N-DHCLV-T-F, N-DHCLV-T-R) (10. mu. mol/L) 0.2. mu.L, Probe (N-DHCLV-T-P) (5. mu. mol/L) 0.5. mu.L, template 1. mu.L, and water to a final volume of 20. mu.L.
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 40 s; 95 ℃ for 5s, 60 ℃ for 25 s, 40 cycles.
Advantageous effects
The invention adopts the primer and the probe for the real-time fluorescent quantitative PCR detection of the N-DHCLV to carry out the detection of the N-DHCLV, 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. The nucleic acid extraction and result judgment only need 100min, and 96 sample detections can be simultaneously carried out at one time.
2. The quantification is accurate: by preparing a standard substance and drawing a standard curve, the infection of the N-DHCLV is directly and accurately quantified according to the Ct value of the N-DHCLV in the sample to be detected.
3. The sensitivity is high: the lowest detectable 55.93 copies/. mu.L.
4. The specificity is strong: the detection of common infectious diseases (such as H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV) in the duck group has no response signal, and only the detection of N-DHCLV has a fluorescence signal.
5. The repeatability is good: the intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method for N-DHCLV detection is 0.69-1.62%, and the inter-group variation coefficient is 0.93-2.41%, which indicates that the established real-time fluorescent quantitative PCR detection method has good repeatability.
Drawings
FIG. 1 amplification curves for real-time fluorescent quantitative PCR, wherein: 1-5: different series of concentrations 5.593X 105—5.593×101Copies/. mu.L template.
FIG. 2 standard curve for real-time fluorescent quantitative PCR.
FIG. 3 sensitivity detection of real-time fluorescent quantitative PCR, in which 1-4: different series of concentrations 5.593X 103-5.593×100Copy/. mu.L template; 5: and (5) negative control.
FIG. 4 specific detection of real-time fluorescent quantitative PCR, wherein: 1: N-DHCLV; c: the test controls (H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV, and MDRV) were not effectively distinguished by the naked eye due to the absence of fluorescent signals.
Detailed Description
The following examples further illustrate the invention.
Example 1
1. Relevant test pathogens
The pathogenic novel duck-origin hepatitis C-like virus (N-DHCLV), duck-origin H9 subtype avian influenza virus (H9-AIV), duck circovirus (DuCV), duck parvovirus (MDPV), duck hepatitis virus types 1 and 3 (DHAV-1, DHAV-3), avian tembusu virus (ATmV) and Muscovy Duck Reovirus (MDRV) for the test are identified and stored by the animal veterinary institute of agricultural academy of sciences of Fujian province.
2. Design and Synthesis of primers and probes
Specific primers (N-DHCLV-T-F and N-DHCLV-T-R) and a probe (N-DHCLV-T-P) of a PCR method for real-time fluorescent quantitative detection of N-DHCLV are designed by referring to the gene sequence characteristics of a novel duck-derived hepatitis C virus (N-DHCLV) and combining the genome characteristics of other hepatitis C virus members, wherein the primers are as follows:
the upstream primer N-DHCLV-T-F: 5'-TGACCCAAACACCAACTTCG-3' the flow of the air in the air conditioner,
the downstream primer N-DHCLV-T-R: 5'-TTCAGTCGCTTCCAATCCAG-3', respectively;
the probe N-DHCLV-T-P is as follows: 5'-AGTCAGAAAATTGTCCCGAGCAGGC-3' the flow of the air in the air conditioner,
the 5 '-end of the fluorescent probe is marked with a fluorescent reporter group FAM, and the 3' -end of the fluorescent probe is marked with a fluorescent quenching group Eclipse.
Primers and probes were synthesized by Baori physician technology (Beijing) Inc.
3. Construction of standard substance of real-time fluorescent quantitative PCR method
According to the sequence characteristics of N-DHCLV (FJ 614 strain) and novel duck-origin hepatitis C virus (N-DHCLV) gene (GenBank accession number MK 737639) in GenBank identified in the early stage of the team, a specific primer is designed by using primer design software Oligo (version v7.37), and the sequence of the primer is as follows: N-DHCLV-F3: 5'-ACCCTGTTTCTGAAGCGAACGT-3' and N-DHCLV-R3: 5'-TCCAGGAACAATTGAAAGGTGT-3', used for amplifying a gene fragment of about 791bp, and the primers were synthesized by Baozi physician's technology (Beijing) Co., Ltd.
Extracting N-DHCLV (FJ 614 strain) nucleic acid RNA by using a virus nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit, removing a first strand of genomic cDNA by using a FastKing one-step method, synthesizing a premixed reagent, carrying out reverse transcription to obtain cDNA, carrying out PCR reaction according to a 2 XTransTaq-T PCR SuperMix (+ dye) instruction, preparing a reaction system by referring to the instruction of the Kit, wherein the reaction system is 50 mu L, 25 mu L of 2 XTransTaq-T PCR SuperMix reaction liquid, 1 mu L of upstream/downstream primers (N-DHCLV-F3 and N-DHCLV-R3, 10 mu M) are respectively used, and the prepared nucleic acid cDNA is 1 mu L, and sterilizing deionized water is supplemented until the final volume is 50 mu L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; 94 ℃ for 50 s, 54 ℃ for 30 s, 72 ℃ for 60 s, 30 cycles; after the circulation is finished, the extension is carried out for 10 min at 72 ℃. And identifying the PCR product by using 1.5% 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 14 h, and then the corresponding plasmid was extracted using fast plasmid mini-extraction Kit. The extracted plasmids are subjected to PCR identification by using primers (N-DHCLV-F3 and N-DHCLV-R3) and conditions during PCR amplification, and the screened positive recombinant plasmids are sent to a doctor-Biotech (Beijing) Limited company for sequencing. And carrying out BLAST analysis verification on the sequencing result on NCBI, wherein the positive recombinant plasmid which is in line with the experimental expectation is used as a positive standard (T-N-DHCLV-T) of the real-time fluorescent quantitative PCR, and the nucleotide homology of the positive recombinant plasmid (the plasmid T-N-DHCLV-T) and the nucleotide homology of the novel duck-origin hepatitis C virus (GenBank accession number MK 737639) are 99.8%.
Measuring the concentration of positive standard (T-N-DHCLV-T) with micro nucleic acid analyzer, and calculating its copy number to be 5.593 × 108Copies/. mu.L, were serially diluted 10-fold and the plasmid contents were 5.593X 10, respectively7～5.593×100Copying/microliter, subpackaging and storing at-20 ℃ for later use.
4. Real-time fluorescent quantitative PCR reaction condition
Taking an N-DHCLV positive standard (T-N-DHCLV-T) as a template, and carrying out real-time fluorescence quantitative PCR reaction at different annealing temperatures (54-64 ℃) and under the concentration (2.5-20 mu mol/L) of a primer (N-DHCLV-T-F, N-DHCLV-T-R) and the concentration (1.25-10 mu mol/L) of a probe (N-DHCLV-T-P), so as to optimize the reaction conditions. 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 N-DHCLV infection if the positive fluorescence signal exists.
The optimal reaction system 20 mul optimized by the established real-time fluorescence quantitative PCR detection method of the N-DHCLV is as follows: PremixEx Taq (Probe qPCR) mixture 10. mu.L, each of upstream and downstream primers (N-DHCLV-T-F, N-DHCLV-T-R) (10. mu. mol/L) 0.2. mu.L, Probe (N-DHCLV-T-P) (5. mu. mol/L) 0.5. mu.L, template 1. mu.L, and water were added to a final volume of 20. mu.L.
The optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 40 s; 95 ℃ for 5s, 60 ℃ for 25 s, 40 cycles.
Using optimized reaction conditions at 5.593X 105—5.593×101Copy/. mu.L was used as template to obtain an amplification kinetics curve (see FIG. 1).
The normal logarithm (lgC) of the plasmid content (C) in each standard substance is taken as an abscissa, the cycle number threshold (Ct value) is taken as an ordinate, and an N-DHCLV real-time fluorescence quantitative PCR standard curve (shown in figure 2) is obtained, wherein the slope of the obtained standard curve is-3.564, the Y-axis intercept is 40.06, the correlation coefficient is 1.000, and the amplification efficiency is 0.91, so that the experimental expectation is met.
5. Sensitivity detection
Using optimized reaction conditions at 5.593X 103—5.593×100Copy/. mu.L is used as template to obtain the lowest detection limit of real-time fluorescence quantitative PCR. As can be seen from FIG. 3, the lowest detection limit of the established real-time fluorescent quantitative PCR method is 5.593X 101Copies/. mu.L (i.e., 55.93 copies/. mu.L).
6. Specificity detection
No response signal was detected for common infectious diseases in duck group (such as H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV), and only fluorescence signal appeared for N-DHCLV detection (FIG. 4).
7. Repeatability test
The intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method for N-DHCLV detection is 0.69-1.62%, and the inter-group variation coefficient is 0.93-2.41%, which indicates that the established real-time fluorescent quantitative PCR detection method has good repeatability.
TABLE 1 determination of coefficient of variation for real-time fluorescent quantitative PCR method
8. Clinical application
After 65 clinically collected duck disease materials are detected by using the established N-DHCLV real-time fluorescent quantitative PCR detection method, 5 parts of N-DHCLV is detected to be infected positively, and the copy numbers are 3.503 multiplied by 10 respectively2Copy/. mu.L, 4.332X 103Copy/. mu.L, 9.413X 103Copies/. mu.L and 8.003X 103Copy/. mu.L, positive rate is 7.69%.
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.
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<213> Artificial sequence
<400>3
agtcagaaaa ttgtcccgag caggc 25
<210>4
<211>22
<212>DNA
<213> Artificial sequence
<400>4
accctgtttc tgaagcgaac gt 22
<210>5
<211>22
<212>DNA
<213> Artificial sequence
<400>5
tccaggaaca attgaaaggt gt 22
Claims (2)
1. A primer and a probe for real-time fluorescent quantitative PCR detection of N-DHCLV are characterized in that: the primer sequences are as follows:
the upstream primer N-DHCLV-T-F: 5'-TGACCCAAACACCAACTTCG-3' the flow of the air in the air conditioner,
the downstream primer N-DHCLV-T-R: 5'-TTCAGTCGCTTCCAATCCAG-3', respectively;
the probe N-DHCLV-T-P is as follows: 5'-AGTCAGAAAATTGTCCCGAGCAGGC-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. An N-DHCLV real-time fluorescent quantitative PCR detection kit is characterized in that: the kit comprises the primer and the probe of claim 1.
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