CN111304371B - Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus wild strain - Google Patents
Locked nucleic acid probe fluorescent quantitative PCR detection composition, detection method and detection kit for African swine fever virus wild strain Download PDFInfo
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Abstract
The invention discloses a locked nucleic acid probe fluorescent quantitative PCR detection composition, a detection method and a detection kit of a wild strain of African swine fever virus. The detection composition disclosed by the invention is designed by utilizing the characteristics of locked nucleic acid, has strong identification capability and affinity to DNA, can effectively improve the detection efficiency of the African swine fever virus wild strain, and reduces the probability of missed detection. The invention provides a new detection method and a detection reagent for identifying and detecting the African swine fever virus wild strain, compared with the existing detection method, the invention has the advantages of strong specificity, high sensitivity and the like, and the sensitivity is 10 times higher than that of the conventional TaqMan probe fluorescent quantitative PCR method; can be used for distinguishing African swine fever virus wild strains and vaccine strains with MGF360-505R gene deletion, is particularly suitable for scientific research and clinical application, and has good commercial application value.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a locked nucleic acid probe fluorescent quantitative PCR detection composition, a detection method and a detection kit for a wild strain of African swine fever virus.
Background
African Swine Fever (ASF) is an acute, virulent and highly contagious infectious disease of domestic and wild pigs caused by African Swine Fever Virus (ASFV), and the death rate of the domestic pigs due to acute infection can reach 100%. The world animal health Organization (OIE) ranks the animal epidemic disease as a legally reported animal epidemic disease, and China ranks the animal epidemic disease as a type of animal epidemic disease.
The patent of the attenuated African swine fever virus with gene deletion and the application thereof as a vaccine (publication number: CN110093324A) discloses that the African swine fever Pig/CN/HLJ/2018 Chinese epidemic strain MGF360-505R gene deletion or the combined deletion of CD2V and MGF360-505R genes can provide 100% immune protection, and the social application value is extremely high. However, after the vaccine is used, the existing conventional detection method cannot distinguish the positive wild virus infection and the positive vaccination. The detection method based on the MGF360-505R gene can be used as a detection method of the African swine fever virus wild strain and is used for distinguishing the African swine fever virus wild strain from a vaccine strain.
The current ASF laboratory detection method mainly comprises virus separation, ELISA, PCR and the like. The virus separation and ELISA are complicated to operate, and the experimental conditions are severe, so that the virus separation and ELISA are difficult to carry out in laboratories with common conditions. Fluorescent quantitative PCR has been widely used in the detection of ASFV, but has a disadvantage of producing false positive results due to non-specific amplification. Therefore, the existing conventional TaqMan probe fluorescent quantitative PCR detection method still needs to be further improved, and the application of an improved probe design strategy becomes a new direction of a nucleic acid detection method.
A Locked Nucleic Acid (LNA) probe selectively modifies some basic groups in the probe into LNA basic groups on the basis of a TaqMan probe, greatly improves the affinity of the probe and a target sequence, and has higher stability, specificity and sensitivity. The MGF360-505R gene is detected by using the nucleic acid probe fluorescent quantitative PCR, so that a more specific and sensitive detection method is provided for the detection of the African swine fever virus wild strain.
Disclosure of Invention
The invention aims to make up the defects of the prior art and provide a locked nucleic acid probe fluorescent quantitative PCR detection composition for a wild strain of African swine fever virus.
The invention also aims to provide a locked nucleic acid probe fluorescent quantitative PCR detection kit of the African swine fever virus wild strain, which comprises the detection composition.
The invention further aims to provide a fluorescence quantitative PCR detection method of the locked nucleic acid probe of the African swine fever virus wild strain based on the detection kit.
The purpose of the invention is realized by the following technical scheme: a locked nucleic acid probe fluorescence quantitative PCR detection composition of a wild strain of African swine fever virus comprises 1 group of specific primer pairs for amplifying the wild strain of African swine fever virus and 1 LNA-Taqman probe in an amplification target region of the primer pairs.
The sequences of the specific primer pairs are shown as Seq ID No.1 and Seq ID No. 2.
The sequence of the LNA-Taqman probe is shown as Seq ID No. 3.
In the sequence of the LNA-Taqman probe, a base 'T' at the 11 th position, a base 'C' at the 12 th position and a base 'C' at the 13 th position are all locked nucleic acids.
The LNA-Taqman probe is marked with a fluorescent substance at the 5 'end and marked with a quenching substance at the 3' end.
The fluorescent substance labeled at the 5' end is labeled FAM.
The 3' end is marked with a quenching substance and is marked with BHQ 1.
The locked nucleic acid probe fluorescent quantitative PCR detection composition of the African swine fever virus wild strain is applied to preparation of a kit for detecting the African swine fever virus wild strain.
A locked nucleic acid probe fluorescence quantitative PCR detection kit of a wild strain of African swine fever virus comprises the detection composition.
The detection kit also comprises a positive control.
The positive control is a recombinant plasmid containing MGF360-505R gene of African swine fever virus wild strain.
The vector of the recombinant plasmid is pUC 57.
The lock nucleic acid probe fluorescent quantitative PCR detection kit of the African swine fever virus wild strain is applied to identification and/or detection of the African swine fever virus wild strain.
A fluorescence quantitative PCR detection method of a locked nucleic acid probe of a wild strain of African swine fever virus comprises the following steps:
(1) extracting genomic DNA of a sample;
(2) preparing a nucleic acid locking probe fluorescent quantitative PCR reaction system;
(3) placing the locked nucleic acid probe fluorescent quantitative PCR reaction system prepared in the step (2) into a fluorescent quantitative PCR instrument for amplification reaction;
(4) and (3) comparing the cycle threshold Ct of the genome DNA with the standard curve to obtain the copy concentration of the African swine fever virus gene fragment in the genome DNA.
The locked nucleic acid probe fluorescent quantitative PCR reaction system in the step (2) is as follows: premix Ex Tag (2X) 10. mu.L, upstream primer 0.4. mu.L at a concentration of 10. mu. mol/L (concentration of 0.2. mu. mol/L in the system), downstream primer 0.4. mu.L at a concentration of 10. mu. mol/L (concentration of 0.2. mu. mol/L in the system), probe 0.2. mu.L at a concentration of 10. mu. mol/L (concentration of 0.1. mu. mol/L in the system), template DNA 1. mu.L, and sterilized water to 20. mu.L.
The sequences of the upstream primer, the downstream primer and the probe are shown as Seq ID No.1, Seq ID No.2 and Seq ID No.3 in sequence.
The reaction conditions of the amplification reaction in the step (3) are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 15s, 60 ℃ for 30s, for 45 cycles.
Compared with the prior art, the invention has the following advantages and effects:
(1) the detection composition for the African swine fever virus wild strain is designed by utilizing the characteristic of locked nucleic acid, has strong recognition capability and affinity to DNA, can effectively improve the detection efficiency of the African swine fever virus wild strain, and reduces the probability of missed detection.
(2) Compared with the existing detection method, the detection composition, the detection kit and the detection method have the advantages of strong specificity, high sensitivity and the like, are 10 times higher than the sensitivity of the conventional TaqMan probe fluorescent quantitative PCR method, are suitable for scientific research and clinical application, and have good commercial application value.
(3) The African swine fever virus wild strain detection composition, the detection kit and the detection method based on the MGF360-505R gene can be used for distinguishing the African swine fever virus wild strain from a vaccine strain with MGF360-505R gene deletion.
Drawings
FIG. 1 is a graph showing the results of sensitivity detection of LNA TaqMan probe and TaqMan probe fluorescent quantitative PCR; wherein A is an LNA probe amplification curve; b is a TaqMan probe amplification curve; 1 is 5.7X 107Copy, 2 is 5.7X 106Copy, 3 is 5.7X 105Copy, 4 is 5.7X 104Copy, 5 is 5.7X 103Copy, 6 is 5.7X 102Copy, 7 is 5.7X 101Copy, 8 is 5.7X 100And (6) copying.
FIG. 2 is a graph showing the results of specificity detection; wherein 1 is African swine fever virus positive recombinant plasmid pUC57-ASFV-MGF360-505R (5.7 x 10)5Copy); 2 is hog cholera virus; 3 is porcine circovirus type 2; 4 is porcine pseudorabies virus; and 5 is a negative control.
FIG. 3 shows a positive recombinant plasmid pUC57-ASFV-MGF360-505R (5.7X 10)5Copies) were performed.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and the accompanying drawings, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Materials, methods and results
1. Viral strains
Classical swine fever virus, porcine circovirus type 2 and porcine pseudorabies virus are purchased from commercial vaccines in the market, wherein the classical swine fever virus live vaccine is purchased from Guangdong Yongshu biopharmaceutical corporation, the porcine pseudorabies virus live vaccine (Bartha-K61 strain) and the porcine circovirus type 2 inactivated vaccine are purchased from Shanghai Haili biotechnology Limited.
2. Primary reagents and instruments
2 × Premix Ex Tag (Probe qPCR) (available from TaKaRa, Cat No. RR390). The reagent used in the application is an analytical pure or biochemical reagent, and the experimental water meets the specification of first-grade water in GB/T6682. All reagents were dispensed in containers without dnase contamination. Fluorescence quantitative PCR instruments (LightCycler96), ultraviolet spectrophotometer (BioTek) and the like are all available from the laboratory.
3. Design and screening of primers and probes
Specific primers and probes are designed according to the conserved region sequence of the African swine fever virus MGF360-505R gene. Specific sequences of the primers and probes are shown in Table 1.
TABLE 1 fluorescent quantitative PCR primer and Probe sequences
In Table 1, the 11 th, 12 th and 13 th bases of the F1R1-probe1 probe are locked nucleic acid modified bases; in the F2R2-probe1 probe, the 6 th, 7 th, 8 th and 9 th bases are locked nucleic acid modified bases; in the F3R3-probe1 probe, the 6 th, 7 th and 8 th bases are locked nucleic acid modified bases; all probes were labeled with FAM at the 5 'end and BHQ1 at the 3' end.
4. Preparation of Positive control in kit
The positive control plasmid pUC57-ASFV-MGF360-505R was synthesized by Biotechnology engineering (Shanghai) GmbH. After the sequencing result is compared with the GenBank sequence without errors, the concentration and the purity of the plasmid are determined by adopting a full-automatic ultraviolet spectrophotometer.
5. Viral nucleic acid extraction
Extracting hog cholera virus, porcine circovirus type 2 and porcine pseudorabies virus genomes as templates according to the kit operation instructions.
6. Fluorescent quantitative PCR reaction system and reaction conditions
The reaction system is shown in table 2, and the reaction conditions are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 45 cycles, 15s, 60 ℃ for 30 s. After the amplification is finished, the specificity of the reaction system and the primer thereof is verified.
TABLE 2 Real-time PCR reaction System
When the primer probe combinations in Table 1 were screened by alignment, the positive recombinant plasmid of step 4 was detected in combination 2 (combination of the upstream primer F2 having the sequence shown in Seq ID No.5, the downstream primer R2 having the sequence shown in Seq ID No.6 and the probe F2R2-probe1 having the sequence shown in Seq ID No. 7) and combination 3 (combination of the upstream primer F3 having the sequence shown in Seq ID No.8, the downstream primer R3 having the sequence shown in Seq ID No.9 and the probe F3R3-probe1 having the sequence shown in Seq ID No. 10). Only combination 1 (combination of the upstream primer F1 having a sequence shown by Seq ID No.1, the downstream primer R1 having a sequence shown by Seq ID No.2 and the probe F1R1-probe1 having a sequence shown by Seq ID No. 3) gave the desired amplification result. Finally, an upstream primer F1 with a sequence shown in Seq ID No.1, a downstream primer R1 with a sequence shown in Seq ID No.2 and a probe F1R1-probe1 with a sequence shown in Seq ID No.3 are screened from the primer probes in the table 1, and the primer and probe combination can be used for efficiently detecting wild strains of the African swine fever virus.
7. Establishment of Standard Curve and sensitivity test
The positive recombinant plasmid pUC57-ASFV-MGF360-505R with the concentration and purity determined in the step 4 is diluted by 10 times to obtain 5.7X 100～5.7×107The recombinant plasmids were used as standard templates (numbered 1, 2, 3, 4, 5, 6, 7, 8 in sequence) at 8 dilutions per copies/. mu.L, with 3 replicates per template concentration. And (3) amplifying LNA TaqMan probe fluorescent quantitative PCR (an upstream primer shown by SEQ ID No.1, a downstream primer shown by SEQ ID No.2 and a probe shown by SEQ ID No. 3) and conventional TaqMan probe fluorescent quantitative PCR (an upstream primer shown by SEQ ID No.1, a downstream primer shown by SEQ ID No.2 and a probe shown by SEQ ID No. 4) according to the reaction system and the reaction parameters of the fluorescent quantitative PCR established in the step (6), obtaining a fluorescent amplification curve and drawing a standard curve. And determining the lowest copy number of the detected recombinant plasmid by observing an amplification curve, and finally establishing a standard curve by taking the Ct value as a vertical coordinate and the logarithm of the copy number as a horizontal coordinate to evaluate the sensitivity of the whole PCR system.
As a result, pUC57-ASFV-MGF 360-505R-positive recombinant plasmid was diluted 10-fold to 5.7X 100～5.7×1078 dilutions of copies/. mu.L, LNA TaqMan probe for fluorescent quantitative PCR amplification to obtain amplification curves, as shown in part A of FIG. 1, the concentration of standard curve is 5.7X 100～5.7×107The correlation is very good in the range of copies/. mu.L, and the linear equation y is-3.461 x +40.909, R20.9942, drawing a standard curve; performing conventional TaqMan probe fluorescent quantitative PCR amplification to obtain an amplification curve, as shown in part B of FIG. 1, the concentration of the standard curve is 5.7 × 101～5.7×107The correlation is very good in the range of copies/. mu.L, and the linear equation y is-3.6879 x +44.444, R2A standard curve is plotted, 0.9923.
The detection result of taking the recombinant plasmids with different copy numbers as a template shows that the amplification curve of the gene presents a typical S shape and the intervals of all the curves are uniform. The minimum detection amount of the MGF360-505R gene based LNA TaqMan probe fluorescent quantitative PCR method is 5.7 copies, the minimum detection amount of the conventional TaqMan probe fluorescent quantitative PCR method is 57 copies, and the sensitivity of the LNA TaqMan probe fluorescent quantitative PCR method is 10 times higher than that of the conventional TaqMan probe fluorescent quantitative PCR method.
8. Specificity detection
Adopting the fluorescent quantitative PCR reaction system and reaction parameters established in the step 6, and the reaction rate is 5.7 multiplied by 105The copied positive plasmid pUC57-ASFV-MGF360-505R, classical swine fever virus, porcine circovirus type 2, and porcine pseudorabies virus genome are used as templates, and sterile water is used as negative control; the specificity of the established method was verified by performing locked nucleic acid probe fluorescent quantitative PCR amplification using a roche LightCycler480 fluorescent quantitative PCR instrument.
As shown in FIG. 2, only pUC57-ASFV-MGF360-505R positive recombinant plasmid produced a specific fluorescence curve, and the others were negative, which proved that the method had better specificity.
9. Repeatability detection
Using 5.7X 105The copied positive plasmid pUC57-ASFV-MGF360-505R is taken as a template, and the locked nucleic acid probe is subjected to fluorescence fixation according to the step 6The stability was analyzed by measuring PCR assay and repeating the assay three times. The results are shown in FIG. 3, the detection results of the curves 1 to 3 are basically consistent, and corresponding fluorescence curves can be observed at the same position, which indicates that the locked nucleic acid probe fluorescence quantitative PCR detection method has good repeatability.
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
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Claims (10)
1. A locked nucleic acid probe fluorescent quantitative PCR detection composition of a wild strain of African swine fever virus is characterized by comprising 1 group of specific primer pairs for amplifying the wild strain of African swine fever virus and 1 LNA-Taqman probe:
the sequences of the specific primer pair are shown as Seq ID No.1 and Seq ID No. 2;
the sequence of the LNA-Taqman probe is shown as Seq ID No. 3;
the 11 th, 12 th and 13 th bases of the LNA-Taqman probe are locked nucleic acid modified bases.
2. The fluorescence quantitative PCR detection composition for the locked nucleic acid probe of the African swine fever virus wild strain according to claim 1, which is characterized in that: the LNA-Taqman probe is marked with a fluorescent substance at the 5 'end and marked with a quenching substance at the 3' end.
3. The fluorescence quantitative PCR detection composition for the locked nucleic acid probe of the African swine fever virus wild strain according to claim 2, which is characterized in that: the 5 'end mark fluorescent substance is marked FAM, and the 3' end mark quenching substance is marked BHQ 1.
4. Use of the locked nucleic acid probe fluorescent quantitative PCR detection composition of the African swine fever virus wild strain according to any one of claims 1-3 in preparation of a detection kit for the African swine fever virus wild strain.
5. A locked nucleic acid probe fluorescent quantitative PCR detection kit of a wild strain of African swine fever virus, which is characterized by comprising the locked nucleic acid probe fluorescent quantitative PCR detection composition of the wild strain of African swine fever virus of any one of claims 1 to 3.
6. The fluorescence quantitative PCR detection kit for locked nucleic acid probe of African swine fever virus wild strain according to claim 5, characterized in that:
the detection kit also comprises a positive control;
the positive control is a recombinant plasmid containing MGF360-505R gene of African swine fever virus wild strain.
7. The fluorescence quantitative PCR detection kit for locked nucleic acid probe of African swine fever virus wild strain according to claim 6, characterized in that: the vector of the recombinant plasmid is pUC 57.
8. Use of the locked nucleic acid probe fluorescent quantitative PCR detection kit for a wild strain of African swine fever virus according to claim 5, 6 or 7 for the identification and/or detection of a wild strain of African swine fever virus for non-disease diagnostic purposes.
9. A fluorescence quantitative PCR detection method of locked nucleic acid probe of African swine fever virus wild strain is a non-disease diagnosis purpose method, and is characterized by comprising the following steps:
(1) extracting genomic DNA of a sample;
(2) preparing a nucleic acid locking probe fluorescent quantitative PCR reaction system;
(3) placing the locked nucleic acid probe fluorescent quantitative PCR reaction system prepared in the step (2) into a fluorescent quantitative PCR instrument for amplification reaction;
(4) comparing the cycle threshold Ct of the genome DNA with a standard curve to obtain the copy concentration of the African swine fever virus gene fragment in the genome DNA;
wherein the sequences of the upstream primer, the downstream primer and the probe are shown as Seq ID No.1, Seq ID No.2 and Seq ID No.3 in sequence; the 11 th, 12 th and 13 th bases of the probe are locked nucleic acid modified bases.
10. The fluorescence quantitative PCR detection method for the locked nucleic acid probe of the African swine fever virus wild strain according to claim 9, which is characterized in that:
the locked nucleic acid probe fluorescent quantitative PCR reaction system in the step (2) is as follows: 2 XPremix Ex Tag 10. mu.L, upstream primer 0.4. mu.L at a concentration of 10. mu. mol/L, downstream primer 0.4. mu.L at a concentration of 10. mu. mol/L, probe 0.2. mu.L at a concentration of 10. mu. mol/L, template DNA 1. mu.L, and sterilized water to 20. mu.L;
the reaction conditions of the amplification reaction in the step (3) are as follows: pre-denaturation at 95 deg.C for 10 min; amplification was carried out at 95 ℃ for 15s, 60 ℃ for 30s, for 45 cycles.
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| CN113817727A (en) * | 2021-11-22 | 2021-12-21 | 苏州蝌蚪生物技术有限公司 | Amplification primer composition and kit for detecting African swine fever virus |
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