CN111961758B - N-DMV and N-DHCLV dual real-time fluorescent quantitative PCR identification and detection primer and kit - Google Patents
N-DMV and N-DHCLV dual real-time fluorescent quantitative PCR identification and detection primer and kit Download PDFInfo
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Abstract
The invention provides a primer and a kit for dual real-time fluorescent quantitative PCR identification and detection of N-DMV and N-DHCLV, wherein the primer sequence is shown as SEQ ID NO.1-4, and the primer and the kit have high specificity and sensitivity. The established method can simultaneously detect the epidemiological conditions and the infection degree of the N-DMV and the N-DHCLV in the duck group, simplify the operation procedure and save the cost. After the real-time fluorescent quantitative PCR reaction is finished, the result can be directly judged by observing an amplification curve and a Tm value. The establishment of the invention can fill the blank of related fields at home and abroad.
Description
Technical Field
The invention provides a novel duck-origin Megri virus (N-DMV) and novel duck-origin hepatitis C-like virus (N-DHCLV) dual real-time fluorescence quantitative PCR detection primer and a kit, belonging to the field of zoonosis.
Background
The Real-time fluorescent quantitative PCR method (Real time PCR) is a method for detecting the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemicals in a DNA amplification reaction. A method for quantitatively analyzing a specific DNA sequence in a sample to be tested by an internal reference (or an external reference method). Because of the exponential phase of the PCR amplification, there is a linear relationship between the Ct value of the template and the initial copy number of the template. The real-time fluorescence quantitative PCR is to detect the PCR process in real time through a fluorescence signal in the PCR amplification process. The real-time fluorescent quantitative PCR technology can not only qualitatively detect the existence of pathogen, but also quantitatively analyze the virus content when detecting the pathogen, and is widely used in the pathogen nucleic acid detection technology. The double real-time fluorescent quantitative PCR is a special fluorescent quantitative PCR form, and has the outstanding characteristics that two pathogens can be simultaneously detected by one real-time fluorescent quantitative PCR reaction, and the method is very effective for identifying and detecting pathogens with complex pathogens or multiple genotypes.
Family of picornaviridae (A)Picornaviridae) Viruses are a spherical, non-enveloped, single-stranded, plus-strand virus with a particle diameter of about 20-30 nm. Early genomics studies found that picornaviridae viruses were generally 6.6kb to 9.8kb in length. The genome of a typical picornaviridae virus typically contains only one large Open Reading Frame (ORF), flanked by 5 '-UTR and 3' -UTR, respectively. At present, members of the genus Megrivirus (the Chinese name is temporarily unidentified) of the family of turkey, chicken, pigeon and duck-derived picornaceae have been found. A genome analysis of a novel duck-origin Megri virus (novel duck-origin Megri virus, N-DMV) and a novel duck-origin Megri virus (novel duck-origin Megri virus, N-DMV) (Liao Q, et al. Genomic characterization of a novel picornavir in Pekin viruses 2014, 172(1-2):78-91. doi: 10.1016/j. vetmic.2014.05.002.) finds that the N-DMV has a genome structure similar to that of viruses of the Megrivirus genus, and a polyprotein of the N-DMV contains characteristic motifs of typical picornaviridae viruses but has characteristics of the N-DMV. The N-DMV genome, excluding the poly (A) tail, is 9700bp in length, the longest in the family of microviviridae. The most prominent feature is located in the 2A region, which consists of two proteins of unknown function (2A 1, 2A 2) and a parachovirus-like 2A 3. The research also finds that the N-DMV has similar genome structural characteristics with the turkey-derived, chicken-derived and pigeon-derived Megri viruses and is inheritedAnd the cells are in the same genetic evolutionary branch.
Flaviviridae (Flaviviridae) is a small enveloped virus family with a single positive stranded RNA genome, in which most viruses infect mammals and birds, and many viruses are host-specific and pathogenic. Phylogenetic relationships based on the amino acid sequences of the conserved regions of RdRP indicate that members of the flaviviridae family are clustered among the 4 currently designated genera of viruses, including flaviviruses (flaviviruses), pestiviruses (pestiviruses), hepaciviruses (hepaciviruses), and pageviruses (pegiviruses). Among them, tamara bat virus (Tamana bat virus) is distributed in a separate branch, tentatively listed as a potential member of the Flavivirus genus (Flavivirus). 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-origin hepatitis C-like virus (N-DHCLV) (non-duck hepatitis virus-like, N-DHCLV) (Chu L, et al. A high stem di-vergent hepatitis virus-like virus in home products. J Gen Virus. 2019, 100(8):1234-1240. doi: 10.1099/jgV.0.001298) is a newly discovered HCV-homologous virus (HCV-like virus) that has been discovered in the egg-laying genetic duck group in recent years. 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 hepatitis C virus-like virus members.
Currently, there are two new pathogens in duck populations-a novel duck-derived Megri virus (N-DMV) and a novel duck-derived hepatitis c-like virus (N-DHCLV). Therefore, the double real-time fluorescent quantitative PCR detection method capable of simultaneously detecting the N-DMV and the N-DHCLV is established, not only can simultaneously detect two new epidemic diseases prevalent in duck groups, but also can simplify the operation procedure, save the cost and provide technical support for the subsequent molecular epidemiological investigation and pathogenic mechanism research of related pathogens. However, at present, no research report of a double real-time fluorescent quantitative PCR detection kit for simultaneously detecting N-DMV and N-DHCLV is found at home and abroad, and the establishment of the invention can fill the blank of research in related fields at home and abroad.
Disclosure of Invention
The invention aims to provide a primer and a kit for dual real-time fluorescent quantitative PCR (polymerase chain reaction) identification and detection of N-DMV and N-DHCLV, the method has the advantages of high sensitivity, good stability, strong specificity and good repeatability, and the problem that no relevant research report of dual real-time fluorescent quantitative PCR detection for simultaneously detecting N-DMV and N-DHCLV exists at present is solved.
In order to realize the purpose, the following technical scheme is adopted:
the dual real-time fluorescent quantitative PCR identification detection primers of the N-DMV and the N-DHCLV comprise the following two groups of primers:
for detection of N-DMV:
the upstream primer N-DMV-q2SYF 1: 5'-ATATATAAGGATGGCTGGTGCA-3' the flow of the air in the air conditioner,
downstream primer N-DMV-q2SYR 1: 5'-CATTCATCACACCTACAGATCCAT-3' the flow of the air in the air conditioner,
the size of the target fragment is 131 bp;
for detecting N-DHCLV:
the upstream primer N-DHCLV-q2SYF 1: 5'-TTGTGGCCATTCTGTCATACTC-3', and the adhesive tape is used for adhering the film to a substrate,
downstream primer N-DHCLV-q2SYR 1: 5'-AGGAGTACTAAACCACCCAGATC-3' the flow of the air in the air conditioner,
the size of the target fragment was 154 bp.
The N-DMV and N-DHCLV double real-time fluorescence quantitative PCR detection kit comprises the primer.
And (5) judging a result:
after the real-time fluorescent quantitative PCR reaction is finished, observing the analysis test result of the peak value (Tm value) of the dissolution curve: if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (83.09 +/-0.10) ° C, judging that the N-DMV is positive; if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (86.95 +/-0.08) ° C, judging that the N-DHCLV is positive; if both Tm = (83.09. + -. 0.10) ° C and Tm = (86.95. + -. 0.08) ° C, a peak appears, indicating mixed infection of N-DMV and N-DHCLV.
The invention provides a primer and a kit for detecting N-DMV and N-DHCLV by double real-time fluorescence quantitative PCR, which have the following advantages and effects:
1.N-DMV and N-DHCLV can be detected simultaneously.
2. 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 100 min, and 96 sample detections can be simultaneously carried out at one time.
3. The quantification is accurate: by preparing a standard substance and drawing a standard curve, the infection of N-DMV and N-DHCLV can be directly and accurately quantified according to the Ct value of the N-DMV and the N-DHCLV in a sample to be detected.
4. The sensitivity is high: the lowest detectable 77.58 copies/μ L of N-DMV; the lowest 49.23 copies/. mu.L of N-DHCLV can be detected.
5. The specificity is strong: no response signal is detected on common infectious diseases (such as H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV) in the duck group, only fluorescence signals appear on N-DMV and N-DHCLV detection, and the difference of specific dissolution curve peak values (Tm values) exists.
Drawings
FIG. 1 amplification curve of N-DMV real-time fluorescent quantitative PCR, wherein: 1-4: different series of concentrations 7.758X 10 4 ～7.758×10 1 Copy/. mu.L template.
FIG. 2 standard curve of N-DMV real-time fluorescent quantitative PCR.
FIG. 3 detection of sensitivity of N-DMV real-time fluorescent quantitative PCR.
FIG. 4 specific detection of N-DMV real-time fluorescent quantitative PCR, wherein: 1: N-DMV; c: the test controls (e.g., N-DHCLV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV, and MDRV) were indistinguishable to the naked eye due to no fluorescence signal.
FIG. 5 melting curve analysis of N-DMV real-time fluorescent quantitative PCR, in which: 1: N-DMV; c: the test controls (e.g., N-DHCLV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV, and MDRV) were visually indistinguishable due to no fluorescent signal.
FIG. 6 amplification curves for N-DHCLV real-time fluorescent quantitative PCR, in which: 1-4: different series of concentrations 4.293X 10 6 ～4.293×10 3 Copies/. mu.L template.
FIG. 7 standard curve of real-time fluorescent quantitative PCR of N-DHCLV.
FIG. 8 detection of sensitivity of N-DHCLV real-time fluorescent quantitative PCR, wherein: 1-3: different series of concentrations 4.293X 10 2 ～4.293×10 0 Copies/. mu.L template.
FIG. 9 specific detection of N-DHCLV real-time fluorescent quantitative PCR, wherein: 1: N-DHCLV; c: the test controls (e.g., N-DMV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV, and MDRV) were visually indistinguishable due to no fluorescent signal.
FIG. 10 melting curve analysis of N-DHCLV real-time fluorescent quantitative PCR, in which: 1: N-DHCLV; c: the test controls (e.g., N-DMV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV, and MDRV) were visually indistinguishable due to no fluorescent signal.
FIG. 11 melting curve analysis for dual real-time fluorescent quantitative PCR, wherein: 1: positive for N-DMV; 2: N-DHCLV positive; 3: N-DMV and N-DHCLV are mixed positive; 4: and (4) negative samples.
Detailed Description
The following examples further describe the invention.
Example 1
1 related test strains
The pathogenic duck novel picornavirus (N-DMV), the novel duck-derived hepatitis C-like virus (N-DHCLV), the duck-derived H9 subtype avian influenza virus (H9-AIV), the duck circovirus (DuCV), the duck parvovirus (MDPV), the duck hepatitis viruses 1 and 3 (DHAV-1 and DHAV-3), the avian tembusu virus (ATmV) and the Muscovy Duck Reovirus (MDRV) for the test are identified and stored by a livestock veterinary institute of agricultural academy of sciences of Fujian province.
2 primer design
According to the genome sequences of novel duck picornavirus (N-DMV) and novel duck-origin hepatitis C virus (N-DHCLV) retrieved from GenBank, specific primers aiming at the N-DMV and the N-DHCLV are designed, and the sequences are as follows:
for detection of N-DMV:
the upstream primer N-DMV-q2SYF 1: 5'-ATATATAAGGATGGCTGGTGCA-3' the flow of the air in the air conditioner,
downstream primer N-DMV-q2SYR 1: 5'-CATTCATCACACCTACAGATCCAT-3' the flow of the air in the air conditioner,
the size of the target fragment is 131 bp.
For detection of N-DHCLV:
the upstream primer N-DHCLV-q2SYF 1: 5'-TTGTGGCCATTCTGTCATACTC-3' the flow of the air in the air conditioner,
downstream primer N-DHCLV-q2SYR 1: 5'-AGGAGTACTAAACCACCCAGATC-3' the flow of the air in the air conditioner,
the size of the target fragment is 154 bp.
Although the length of 131bp and 154 bp is different, the conventional PCR agarose gel electrophoresis cannot effectively distinguish, but the Tm peak value difference exists when the PCR is used for dual real-time fluorescence PCR, so that the PCR can be effectively distinguished.
The primers were all synthesized by Biotechnology engineering (Shanghai) GmbH.
3 nucleic acid extraction and cDNA Synthesis
The nucleic acid RNAs of N-DMV (FJ 785 strain), N-DHCLV (FJ 614 strain), H9-AIV, DHAV-1, DHAV-3, ATmV and MDRV were extracted using the easy pure Viral DNA/RNA Kit from Beijing Quanjin Biotechnology Ltd, according to the instructions. The extracted nucleic acid RNA is reversely transcribed into cDNA by utilizing a FastKing one-step method to remove the first strand synthesis premixed reagent of the genomic cDNA. All are stored at-20 ℃ for later use.
And nucleic acid DNAs (DuCV and MDPV, without reverse transcription) of test controls were extracted simultaneously according to the kit method, and both were stored at-20 ℃ for further use.
Establishment of dual real-time fluorescent quantitative PCR detection method for 4N-DMV and N-DHCLV
4.1 establishment of real-time fluorescent quantitative PCR detection method of N-DMV
4.1.1 preparation of Positive Standard
According to the genome characteristics of the N-DMV (FJ 785 strain), a specific primer is designed by utilizing primer design software Oligo (version v7.37), and the sequence of the primer is as follows: N-DMV-F33: 5'-TGGGAAGAAGTTGAAGAAATGGAC-3' and N-DMV-R33: 5'-AGAGCCAGGTGATATAGCGAGT-3', used for amplifying about 769bp gene fragment. A PCR reaction was carried out using cDNA prepared from N-DMV (strain FJ 785) as a template according to the instructions of 2 XTaq-T PCR Supermix (+ dye), and the reaction system was prepared according to the instructions of the kit, wherein the reaction system was 50. mu.L, 25. mu.L of the 2 XTaq-T PCR Supermix reaction solution, 1. mu.L of each of the upstream and downstream primers (N-DMV-F33 and N-DMV-R33, 10. mu.M), 1. mu.L of the prepared nucleic acid cDNA, and sterile deionized water was added to a final volume of 50. mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; 94 ℃ for 50 s, 55 ℃ for 30s, 72 ℃ for 60s, 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. Cloning the target gene fragment onto a pEASY-T1 vector according to the pEASY-T1 Simple Cloning Kit instruction, randomly selecting 8 single colonies, culturing the single colonies in an ampicillin (the content is 100 mu g/mL) resistant LB liquid culture medium for 14 h, and then extracting corresponding plasmids by using a rapid plasmid miniextraction Kit. PCR identification is carried out on the extracted plasmids by adopting primers (N-DMV-F33 and N-DMV-R33) and conditions during amplification, and the screened positive recombinant plasmids are sequenced by doctor of Hei technology (Beijing) Limited company. The sequencing result is verified by BLAST analysis on NCBI, and the positive recombinant plasmid which is in line with the experimental expectation is used as a positive standard (T-N-DMV-T04) of real-time fluorescence quantitative PCR, and the nucleotide homology of the positive recombinant plasmid (plasmid T-N-DMV-T04) and the nucleotide homology of the novel duck-derived Megri virus (GenBank accession No. KC 663628) are 99.9%.
Measuring the concentration of positive standard (T-N-DMV-T04) with a trace nucleic acid analyzer, and calculating to obtain 7.758 × 10 copy number 8 Copies/. mu.L, were serially diluted 10-fold and the plasmid contents were 7.758X 10, respectively 7 ～7.758×10 0 Copying/mu L, subpackaging and storing at-20 ℃ for later use.
4.1.2 optimization of real-time fluorescent quantitative PCR reaction conditions and establishment of Standard Curve
Taking an N-DMV positive standard (plasmid T-N-DMV-T04) as a template, and carrying out real-time fluorescent quantitative PCR reaction at different annealing temperatures (54-64 ℃) and concentrations (2.5-20 mu mol/L) of primers (N-DMV-q 2SYF1 and N-DMV-q2SYR 1) to optimize reaction conditions.
The optimal reaction system (20. mu.L) is optimized as follows: 10.0. mu.L of TBGreen Mix, 0.4. mu.L each of upstream and downstream primers (N-DMV-q 2SYF1, N-DMV-q2SYR 1) (10 pmol), 2. mu.L of template, and a total volume of 20. mu.L of sterilized double distilled water.
The optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 60 s; at 95 ℃ for 10 s, at 58 ℃ for 25s and at 72 ℃ for 30s, for a total of 40 cycles. After the reaction is finished, a melting curve is made.
And (4) judging a result: when a positive amplification signal is detected, and a single specific peak appears at a melting curve peak value Tm = (83.09 +/-0.10) DEG C, the signal is judged to be N-DMV positive.
Using optimized reaction conditions at 7.758X 10 4 ～7.758×10 1 Copy/. mu.L was used as template to obtain an amplification kinetics curve (see FIG. 1).
The common logarithm of the copy number (lgC) in each concentration standard sample template is used as an abscissa, the cycle number threshold (Ct value) is used as an ordinate, and a linear equation of an N-DMV real-time fluorescence quantitative PCR standard curve (shown in figure 2) is obtained as y = -3.260 x +38.30, the correlation coefficient is 0.986, the amplification efficiency is 99%, which indicates that the established standard curve of the real-time fluorescence quantitative PCR method has a good linear relation.
4.1.3 sensitive assays
Using optimized reaction conditions at 7.758X 10 3 ～7.758×10 0 Copy/. 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 7.758X 10 1 Copies/. mu.L (i.e., 77.58 copies/. mu.L).
4.1.4 specific assays
No response signal was detected for common infectious diseases in duck group (such as N-DHCLV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV), and only fluorescence signal appeared for N-DMV (FIG. 4).
As can be seen from the melting curve in fig. 5, only a single specific peak of N-DMV appears at Tm = (83.09 ± 0.10) ° c. No specific dissolution curve peak is seen for other pathogens (such as N-DHCLV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV), and the established real-time fluorescent quantitative PCR method is strong in specificity.
4.1.5 repeatability test
The intra-group variation coefficient of the established real-time fluorescence quantitative PCR detection method for N-DMV detection is 0.55-1.08%, and the inter-group variation coefficient is 0.61-2.02% (see Table 1), which indicates that the established real-time fluorescence quantitative PCR detection method has good repeatability.
TABLE 1 measurement of coefficient of variation of N-DMV real-time fluorescent quantitative PCR method
4.2 establishment of real-time fluorescent quantitative PCR detection method of N-DHCLV
4.2.1 preparation of Positive Standard
According to the genome characteristics of the N-DHCLV (FJ 614 strain), 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-F44: 5'-ATGCAACTGTAGCTGTTTAGA-3' and N-DHCLV-R44: 5'-AAACTGCCAAACGAAATGT-3', used for amplifying the gene fragment of about 549 bp. A PCR reaction is carried out by using cDNA prepared from N-DHCLV (FJ 614 strain) as a template according to the specification of 2 XTransTaq-T PCR Supermix (+ dye), the reaction system is prepared by referring to the specification of a kit, the reaction system is 50 mu L, wherein 25 mu L of 2 XTransTaq-T PCR Supermix reaction solution and 1 mu L of each of upstream/downstream primers (N-DHCLV-F44 and N-DHCLV-R44, 10 mu M) are used, the prepared nucleic acid cDNA is 1 mu L, and sterile deionized water is supplemented to the final volume of 50 mu L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 52 ℃, 45 s at 72 ℃ and 35 cycles; after the circulation is finished, the extension is carried out for 10 min at 72 ℃. And identifying the PCR product by using 1.5% agarose gel electrophoresis, and cutting and recovering the specific target fragment by using an agarose gel recovery kit. Cloning the target gene fragment onto a pEASY-T1 vector according to the pEASY-T1 Simple Cloning Kit instruction, randomly selecting 8 single colonies, culturing the single colonies in an ampicillin (the content is 100 mu g/mL) resistant LB liquid culture medium for 14 h, and then extracting corresponding plasmids by using a rapid plasmid miniextraction Kit. PCR identification is carried out on the extracted plasmids by using primers (N-DHCLV-F44 and N-DHCLV-R44) and conditions during amplification, and the screened positive recombinant plasmids are sent to Taobaozi physician technology (Beijing) Co. And carrying out BLAST analysis verification on the sequencing result on NCBI, and taking the positive recombinant plasmid which is in line with the experimental expectation as a positive standard (T-N-DHCLV-T04) of the real-time fluorescence quantitative PCR, wherein the nucleotide homology of the positive recombinant plasmid (the plasmid T-N-DHCLV-T04) and the nucleotide homology of the novel duck-origin hepatitis C virus (GenBank accession No. MK 737639) are 100%.
Measuring the concentration of positive standard (T-N-DHCLV-T04) with trace nucleic acid analyzer, and calculating to obtain 4.293 × 10 copy number 9 Copies/. mu.L, were serially diluted 10-fold and the plasmid contents were 4.293X 10, respectively 8 ～4.293×10 0 Copying/microliter, subpackaging and storing at-20 ℃ for later use.
4.2.2 optimization of real-time fluorescent quantitative PCR reaction conditions and establishment of Standard Curve
A DHCLV positive standard (plasmid T-N-DHCLV-T04) is used as a template, and real-time fluorescent quantitative PCR reaction is carried out at different annealing temperatures (54-64 ℃) and concentrations (2.5-20 mu mol/L) of primers (N-DHCLV-q 2SYF1 and N-DHCLV-q2SYR 1), so that the reaction conditions are optimized.
The optimal reaction system (20. mu.L) is optimized as follows: 10.0. mu.L of TBGreen Mix, 0.4. mu.L each of upstream and downstream primers (N-DHCLV-q 2SYF1, N-DHCLV-q2SYR 1) (10 pmol), 2. mu.L of template, and a total of 20. mu.L of the mixture with sterile double distilled water.
The optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 60 s; 10 s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃ for 40 cycles. After the reaction is finished, a melting curve is made.
And (4) judging a result: when a positive amplification signal is detected, and a single specific peak appears at a melting curve peak value Tm = (86.95 +/-0.08) ° C, the result is judged to be N-DHCLV positive.
Using optimized reaction conditions at 4.293X 10 6 ～4.293×10 3 Copy/. mu.L was used as template to obtain an amplification kinetics curve (see FIG. 6).
The common logarithm of the copy number (lgC) in each concentration standard template is used as an abscissa, the threshold value of the cycle number (Ct value) is used as an ordinate, the linear equation of the N-DHCLV real-time fluorescence quantitative PCR standard curve (shown in figure 7) is y = -3.34 x +37.63, the correlation coefficient is 0.999, the amplification efficiency is 98%, and the established standard curve of the real-time fluorescence quantitative PCR method has a good linear relation.
4.2.3 sensitive assays
Using optimized reaction conditions at 4.293X 10 2 ～4.293×10 0 Copy/. mu.L is used as template to obtain the lowest detection limit of real-time fluorescence quantitative PCR. As can be seen from FIG. 8, the lowest detection limit of the established real-time fluorescent quantitative PCR method is 4.293X 10 1 Copies/. mu.L (i.e., 42.93 copies/. mu.L).
4.2.4 specific assays
No response signal is detected on common infectious diseases (such as N-DMV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV) in duck groups, and only a fluorescence signal appears on N-DHCLV detection (figure 9).
As can be seen from the melting curve in fig. 10, only a single specific peak appears for N-DHCLV at Tm = (86.95 ± 0.08) ° c. No specific dissolution curve peak is seen for other pathogens (such as N-DMV, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3, ATmV and MDRV), and the established real-time fluorescence quantitative PCR method is strong in specificity.
4.2.5 repeatability tests
The intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method for N-DHCLV detection is 0.62-1.21%, and the inter-group variation coefficient is 0.68-1.88% (see Table 2), which indicates that the established real-time fluorescent quantitative PCR detection method has good repeatability.
TABLE 2 determination of the coefficient of variation of the N-DHCLV real-time fluorescent quantitative PCR method
4.3 establishment of double EvaGreen real-time fluorescent quantitative PCR detection method
4.3.1 reaction Condition optimization
According to the optimized reaction conditions (mainly aiming at annealing temperature) of 4.1 and 4.2, in order to optimize amplification of the dual real-time fluorescent quantitative PCR reaction, different annealing temperatures (56, 57, 58, 59, 60, 61 and 62 ℃) are selected, the extension time is selected to be 72 ℃ for 30s according to the fragment length, and the optimized conditions are as follows: pre-denaturation at 95 ℃ for 60 s; 10 s at 95 ℃, 30s at 58 ℃ and 30s at 72 ℃ for 40 cycles. After the reaction is finished, a melting curve is made.
4.3.2 primer concentration optimization
The primers for N-DMV (N-DMV-q 2SYF 1/N-DMV-q 2SYR 1) and N-DHCLV (N-DHCLV-q 2SYF1/N-DHCLV-q2SYR 1) were selected for optimization according to the optimized primer concentration conditions of 4.1 and 4.2 by different multiples (8: 1, 4:1, 2:1, 1:2, 1:4, 1: 8). The optimized primers have the effects of 1:1 and 1:2 which are in line with the expectation. In order to save the test cost, the optimized primer is selected to be 1: 1.
4.3.3 optimized conditions
The optimized optimal reaction system (20. mu.L) of the dual real-time fluorescent quantitative PCR method is shown in Table 3 below.
TABLE 3 optimized Dual real-time fluorescent quantitative PCR reaction System
To illustrate, if the clinical sample is detected, 2 μ L of nucleic acid cDNA prepared from the test sample can be directly added.
4.3.4 double real-time fluorescent quantitative PCR method result judgment and specificity analysis
After the real-time fluorescent quantitative PCR reaction was finished, the results of the analysis of the peak value (Tm value) of the dissolution curve were observed (see fig. 11): if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (83.09 +/-0.10) ° C, judging that the N-DMV is positive; if the detection sample has a positive amplification signal and a single specific peak appears at Tm = (86.95 +/-0.08) ° C, judging that the N-DHCLV is positive; if both Tm = (83.09. + -. 0.10) ° C and Tm = (86.95. + -. 0.08) ° C, a peak appears, indicating mixed infection of N-DMV and N-DHCLV.
5 clinical applications
The method comprises the steps of carrying out double real-time fluorescence quantitative PCR detection of N-DMV and N-DHCLV infection on 59 clinical delivery detection duck-origin disease materials, extracting corresponding nucleic acid RNA by using an easy pure 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, and carrying out detection according to the establishment of a double real-time fluorescence quantitative PCR system and conditions. As a result, 7 samples having a single specific peak at Tm = (83.09 +/-0.10) ° C were found, i.e., N-DMV infection was positive, and the positive rate was 11.86%; 5 samples with single specific peaks at Tm = (86.95 +/-0.08) ° C, namely N-DHCLV infection is positive, and the positive rate is 8.47%; and the double peaks (Tm values of 83.09 ℃, 83.10 ℃, 86.96 ℃ and 87.01 ℃) appear in 2 samples, namely the mixed infection of N-DMV and N-DHCLV exists, and the positive rate is 3.39%.
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> animal husbandry and veterinary institute of academy of agricultural sciences in Fujian province
<120> N-DMV and N-DHCLV dual real-time fluorescence quantitative PCR identification detection primer and kit
<130> 8
<160> 8
<170> PatentIn version 3.3
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tgggaagaag ttgaagaaat ggac 24
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aaactgccaa acgaaatgt 19
Claims (2)
- The primer for double real-time fluorescent quantitative PCR identification and detection of N-DMV and N-DHCLV is characterized by comprising the following components in parts by weight: comprises the following two groups of primers:for detection of N-DMV:the upstream primer N-DMV-q2SYF 1: 5'-ATATATAAGGATGGCTGGTGCA-3' the flow of the air in the air conditioner,downstream primer N-DMV-q2SYR 1: 5'-CATTCATCACACCTACAGATCCAT-3' the flow of the air in the air conditioner,the size of the target fragment is 131 bp;for detection of N-DHCLV:the upstream primer N-DHCLV-q2SYF 1: 5'-TTGTGGCCATTCTGTCATACTC-3' the flow of the air in the air conditioner,downstream primer N-DHCLV-q2SYR 1: 5'-AGGAGTACTAAACCACCCAGATC-3' the flow of the air in the air conditioner,the size of the target fragment was 154 bp.
- 2. A N-DMV and N-DHCLV dual real-time fluorescence quantitative PCR detection kit is characterized in that: the kit comprises the primer of claim 1.
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