CN117210615B - TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus - Google Patents
TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus Download PDFInfo
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Abstract
The invention discloses a TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus, and relates to the technical field of molecular biology. The TaqMan fluorescent quantitative RT-PCR detection method of the porcine Mannich virus comprises the following steps: designing and synthesizing primers and probes; synthesizing a virus positive plasmid standard; optimizing an RT-qPCR reaction system; establishing an RT-qPCR standard curve; evaluating an RT-qPCR method; and (5) detecting clinical samples. The TaqMan fluorescent quantitative RT-PCR detection method and application of the porcine Mana corner virus have the advantages of strong specificity, high accuracy, high sensitivity and good repeatability, are suitable for large-scale detection and diagnosis of a pig farm, and have important functions on monitoring and preventing and controlling the porcine Mana corner virus.
Description
Technical Field
The invention relates to the technical field of biological molecular science, in particular to a TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus.
Background
PCR technology, also known as in vitro gene amplification technology, generates PCR products in amountsExponentially increasing, can increase picograms (pg=10) -12 ) Initial test templates of magnitude were amplified to micrograms (μg=10) -6 ) Horizontal. The PCR technology has high specificity and sensitivity, and can detect a target cell from 100 ten thousand cells; in the detection of viruses, the sensitivity of PCR can reach 3 PFU (plaque forming units). PCR technology is mainly used to detect nucleic acids of infectious disease pathogens. Each pathogen has a unique nucleic acid sequence, and detection of specific nucleic acids can identify the pathogen and thus which infectious disease.
The PCR technology has been updated to the third generation since the invention has been 30 years ago, wherein the second generation PCR is the fluorescent quantitative PCR technology (Real-Time PCR), also called qPCR. And the commercial kit for detecting the MenV is mainly divided into a PCR kit and an RT-LAMP isothermal amplification kit, but no approval mark is obtained. Two companies sell the MenV probe method qRT-PCR kit externally, but only one label is used for scientific research, and the rest information is not detailed. In addition, the RT-LAPM isothermal amplification kit and the SYBR Green dye method detection kit have a plurality of problems, compared with PCR, the RT-LAPM detection method has the problems of insufficient detection sensitivity, more difficult primer design (6 pairs of primers are generally required for amplification), high false positive and the like; in view of this, we propose a method for quantitative RT-PCR detection of porcine Mannara Virus (Menangle Virus) TaqMan fluorescence and application thereof.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus, and solves the problems mentioned in the background art.
In order to achieve the above purpose, the invention is realized by the following technical scheme: a method for detecting porcine Mannas carob virus TaqMan fluorescent quantitative RT-PCR, comprising the following steps:
s1: designing and synthesizing a primer and a probe, searching the gene sequence of the porcine Manna corner virus on NCBI, comparing sequences of different strains by utilizing DNAMAN software, and selecting a V-P segment which has high homology and is a genome conserved region as a target gene segment;
s2: synthesizing a virus positive plasmid standard;
s3: optimizing an RT-qPCR reaction system;
s4: establishing an RT-qPCR standard curve;
s5: evaluating an RT-qPCR method, detecting various viruses by adopting the detection method, and recording and comparing experimental results;
s6: and (5) detecting clinical samples.
Optionally, the step S1 further includes: designing probes according to the probe design principle by using Beacon Designer, oligo7 and NCBI BLAST, verifying the probes, and screening the optimal probes; after determining the position of the target probe sequence, primers are designed according to the Primer design principle by utilizing Primer Premier 5, oligo7 and Primer-BLAST software before and after the probe position, and verification is carried out on the primers, so that the optimal Primer pairs are screened.
Optionally, the step S3 includes:
s31: the optimal annealing temperature is screened by a gradient method, and the optimal annealing temperature of the reaction system after optimization is 50 ℃;
s32: the matrix method screens the optimal primer and probe concentrations.
Optionally, the S4 includes: the synthesized positive plasmid is diluted according to 10 times concentration gradient, and RT-qPCR test is carried out by using the optimized system, and a standard curve is drawn.
Optionally, the plurality of viruses includes: porcine circovirus type 2, porcine parvovirus, swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine delta coronavirus, rotavirus, porcine epidemic diarrhea virus.
Optionally, the step S5 includes:
s51: a specificity test; by adopting the detection method to detect various viruses, only the porcine Mannich virus has an amplification curve, and other virus detection results are negative.
S52: susceptibility testing verifies the lowest copy number of the porcine Mana corner virus detected by the method.
S53: repeatability test the reproducibility of the method was verified by repeated measurement of 10-fold diluted V-P plasmid, n=3.
Designing a synthesized primer Probe comprising MenV-p/V-F, menV-p/V-R and MenV-Probe;
the primer sequence of the MenV-p/V-F is 5'-TCCTCGTCATAAAGACAGAA-3'; the primer sequence of the MenV-p/V-R is 5'-GGTGACATATTGGGTTGC-3'; the primer sequence of the MenV-Probe is 5'-FAM-AATAATGTCTATGATGCCGCTCCAATC-BHQ1-3'.
The invention provides a TaqMan fluorescent quantitative RT-PCR detection method and application of porcine Mannich virus. The beneficial effects are as follows:
the porcine Manna corner virus TaqMan fluorescent quantitative RT-PCR detection method and application thereof have the advantages of strong specificity, high accuracy and high sensitivity, can carry out multiple detection, can judge the state of disease infection as early as possible when being applied to disease detection, is suitable for large-scale detection and diagnosis of early infectious diseases, can reliably carry out detection and diagnosis work, and has important functions in monitoring and preventing and controlling porcine Manna corner virus.
Drawings
FIG. 1 is a schematic flow chart of a TaqMan fluorescent quantitative RT-PCR detection method of the porcine Mannich virus of the present invention;
FIG. 2 is a schematic diagram of the design flow of probes and primers of the present invention;
FIG. 3 is a data table of probe and primer sequences of the present invention;
FIG. 4 shows a One Step PrimeScript of the invention TM 20 μl system data sheet recommended by RT-PCR Kit (Perfect Real Time) specification;
FIG. 5 shows a One Step PrimeScript of the invention TM A reaction condition data table using Applied Biosystems7300Real-Time PCR System as recommended by the RT-PCR Kit (Perfect Real Time) specification;
FIG. 6 is a schematic diagram of an amplification curve of an optimal annealing temperature in the optimization of the reaction system of the present invention;
FIG. 7 is a schematic diagram of gel electrophoresis results of optimal annealing temperatures in the optimization of the reaction system of the present invention;
FIG. 8 is a graph showing Ct values at different probe and primer concentrations in the optimization of the reaction system of the present invention;
FIG. 9 is a schematic diagram of the standard curve establishment of the present invention;
FIG. 10 is a schematic diagram of the results of a specificity test of the present invention;
FIG. 11 is a schematic diagram of the result of evaluating sensitivity by the method of the present invention;
FIG. 12 is a schematic diagram of the method of the invention evaluating repeatability results;
FIG. 13 is a schematic diagram showing the results of TaqMan fluorescent quantitative RT-PCR detection of a clinical sample of the porcine Mannich virus of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-13, the present invention provides a technical solution: a method for detecting TaqMan Virus (Menangle Virus) by fluorescence quantitative RT-PCR and application thereof, comprising the following steps:
s1: the design and synthesis of primers and probes, the gene sequences of the porcine Manna corner virus are searched on NCBI, the sequences of different strains are compared by DNAMAN software, and the V-P segment with high homology and a genome conserved region is selected as a target gene segment. Probes were designed and validated according to the probe design rules using Beacon Designer, oligo7, NCBI BLAST. After determining the position of the target probe sequence, primers are designed according to the Primer design principle by using Primer Premier 5, oligo7 and Primer-BLAST software before and after the probe position, and verification is carried out, so that the optimal Primer pair is screened, as shown in figure 3.
S2: and (3) synthesizing a virus positive plasmid standard. Fragments of V-P used to design primers and probes in porcine Mannard Virus (Menangle Virus) were synthesized by general biosystems (Anhui) Inc. as positive plasmid standards for viruses.
S3: optimizing an RT-qPCR reaction system; using One Step PrimeScript TM The 20. Mu.l System recommended by the RT-PCR Kit (Perfect Real Time) specification and the reaction conditions for applying Applied Biosystems7300Real-Time PCR System are shown in FIGS. 4 and 5.
S31: screening the optimal annealing temperature by a gradient method, wherein the data are shown in fig. 6 and 7; the optimal annealing temperature of the reaction system after optimization is 50 ℃.
S32: the matrix method was used to screen for optimal primer and probe concentrations and the data is shown in FIG. 8.
After the reaction system is optimized, the optimal concentration of the upstream/downstream primer is 0.6 mu mol/L; the probe concentration was 0.4. Mu. Mol/L (Ct value=16.66.+ -. 0.15).
S4: the RT-qPCR standard curve is established, and the data are shown in FIG. 9; the synthesized positive plasmid is diluted according to a 10-time concentration gradient, an RT-qPCR test is carried out by using an optimized system, a drawn standard curve is y= -3.4297x+44.4, and the amplification efficiency (E%) is 95.69%, which indicates that the amplification efficiency is high; r is R 2 =0.9999 shows that log values of different concentration gradient template amounts show good correlation with Ct values.
S5: evaluation by RT-qPCR method.
S51: specificity test. As shown in fig. 10; the method is used for detecting porcine circovirus type 2, porcine parvovirus, swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine delta coronavirus, rotavirus and porcine epidemic diarrhea virus which are all negative, and only the porcine Mana horn virus shows an amplification curve, so that the method has high specificity.
S52: sensitivity test. The lowest copy number detectable was 25 copies/. Mu.l, as shown in FIG. 11.
S53: repeatability test. The reproducibility of this method was tested by repeating the measurement on 10-fold dilution of V-P plasmid, n=3, and the result showed CV < 2%, indicating good reproducibility of the method and stable results, as shown in fig. 12.
S6: and (5) detecting clinical samples. Up to now, 720 clinical samples were tested and after the recheck, there were 2 suspected positive samples, as shown in fig. 13.
Designing a synthesized primer Probe comprising MenV-p/V-F, menV-p/V-R and MenV-Probe;
the primer sequence of the MenV-p/V-F is 5'-TCCTCGTCATAAAGACAGAA-3'; the primer sequence of the MenV-p/V-R is 5'-GGTGACATATTGGGTTGC-3'; the primer sequence of the MenV-Probe is 5'-FAM-AATAATGTCTATGATGCCGCTCCAATC-BHQ1-3'.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. A non-diagnostic TaqMan fluorescent quantitative RT-PCR detection method for porcine Mana corner virus is characterized in that: the detection method comprises the following steps:
s1: designing and synthesizing a primer and a probe, searching the gene sequence of the porcine Manna corner virus on NCBI, comparing sequences of different strains on DNAMAN software, and selecting a V-P segment which has high homology and is a genome conserved region as a target gene segment;
s2: synthesizing a virus positive plasmid standard;
s3: optimizing an RT-qPCR reaction system;
s4: establishing an RT-qPCR standard curve;
s5: evaluating an RT-qPCR method;
s6: detecting a clinical sample;
the step S1 further comprises the following steps:
designing probes according to the probe design principle by using Beacon Designer, oligo7 and NCBI BLAST, verifying the probes, and screening the optimal probes;
after determining the position of a target probe sequence, designing primers by utilizing Primer Premier 5, oligo7 and Primer-BLAST software before and after the probe position according to a Primer design principle, verifying the primers, and screening an optimal Primer pair;
the step S3 comprises the following steps:
s31: screening the optimal annealing temperature by a gradient method;
s32: screening the optimal primer and probe concentration by a matrix method;
the synthesized positive plasmid is diluted according to 10 times concentration gradient, the optimized system is used for carrying out RT-qPCR test, and a standard curve is drawn;
the step S5 comprises the following steps:
s51: the specificity test is carried out, the detection method is adopted to detect various viruses, only the porcine Mannich virus has an amplification curve, and other virus detection results except the porcine Mannich virus are negative;
s52: a sensitivity test for verifying the lowest copy number of the porcine Mana corner virus detected by the method;
s53: repeatability test, the repeatability of the method was verified by repeatedly measuring 10-fold diluted V-P plasmid, n=3;
designing a synthesized primer Probe comprising MenV-p/V-F, menV-p/V-R and MenV-Probe;
the primer sequence of the MenV-p/V-F is 5'-TCCTCGTCATAAAGACAGAA-3'; the primer sequence of the MenV-p/V-R is 5'-GGTGACATATTGGGTTGC-3'; the sequence of MenV-Probe is 5'-FAM-AATAATGTCTATGATGCCGCTCCAATC-BHQ1-3'.
2. Use of the non-diagnostic TaqMan fluorescent quantitative RT-PCR assay for porcine mana-keratovirus according to claim 1 for the non-diagnostic detection of porcine mana-keratovirus, wherein the minimum detectable copy number of the TaqMan fluorescent quantitative RT-PCR assay for porcine mana-keratovirus is 25 copies/. Mu.l.
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