CN112746133A - Equine infectious anemia virus fluorescent PCR detection kit and application thereof - Google Patents
Equine infectious anemia virus fluorescent PCR detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses an Equine Infectious Anemia Virus (EIAV) fluorescent PCR detection kit and application thereof. The detection kit contains primers and probes for specifically detecting the equine infectious anemia virus. Compared with the existing detection method, the kit disclosed by the invention can be used for detecting more EIAV strains and has the characteristic of broad spectrum, and only American strain EIAVUK3 and strains with similar sequences can be detected by using the primer probe combination recommended by OIE. The kit has high sensitivity, good specificity and wide strain detection range. The invention provides a more effective technical means for detecting the equine infectious anemia virus.
Description
Technical Field
The invention relates to a PCR detection kit and application thereof, in particular to a fluorescent PCR detection kit for equine infectious anemia virus and application thereof. The invention belongs to the technical field of virus detection.
Background
Equine Infectious Anemia Virus (EIAV) is a member of the lentivirus genus of the family Retroviridae (Retroviridae), and can cause infectious anemia in horses, mules, and donkeys, characterized by fever, anemia, bleeding, jaundice, edema, cardiac dysfunction, blood phase changes, and progressive wasting. According to clinical manifestations, the disease types are classified into 4 types, namely acute, subacute, chronic and recessive, and besides the main symptoms such as fever, anemia, jaundice, bleeding and heart dysfunction, the hematological changes are prominent, such as decrease in red blood cell number, hemoglobin amount, decrease in white blood cell number and appearance of iron-swallowing cells in venous blood. The disease is distributed worldwide, which causes huge economic loss to the horse raising industry. In addition, several cases of infection of the virus have been reported. The sick horse in the fever period is the most dangerous infection source, and the blood and organs (liver, spleen, bone marrow, lymph nodes and the like) contain a large amount of virus and are often discharged out of the body along with secretions and excretions to spread. Chronic horses can be toxic for a long time or even for life. At present, methods for detecting EIAV antigen mainly include PCR method, ELISA method and traditional virus isolation and identification. In contrast, the PCR method has the characteristics of strong specificity, high sensitivity, simple and convenient operation, time saving and the like. The genome of various lentiviruses including EIAV has the characteristic of high variation, and the genome of different strains has great difference, so that the universal quantitative PCR detection method is difficult to establish. The gag gene is a relatively conserved gene sequence in the lentiviral genome, and is therefore usually the gag gene as the primary detection target. The detection method of horse-borne poor fluorescence quantitative PCR recommended by the world animal health Organization (OIE) is designed for the gag gene, but only the American strain EIAV UK3 and the strain with similar sequence can be detected by the method. In recent years, some Asian scholars report that the method cannot detect the strains in China, and the OIE recommended method for the strains in China is also undetectable. Therefore, it is urgently needed to establish a universal, broad-spectrum PCR nucleic acid detection method for equine infectious anemia virus.
Equine infectious anaemia virus members of the lentivirus family, like other retroviruses, form double-stranded viral DNA (commonly referred to as proviral DNA) in the early stages of EIAV infection by the action of viral reverse transcriptase, and the proviral DNA is further integrated into the host chromosome by the action of virally encoded integrase. Therefore, retroviruses including EIAV exist in both RNA and DNA forms in their genome, although they are RNA viruses. Although a large amount of viruses are present in blood and organs of febrile animals, high-speed centrifugation (371000 × g) is required for 2 hours to extract viruses from blood, which takes time and labor; the viscera are not easy to collect, and according to the plan of eliminating the poor sex of horses in China, the positive animals can be killed and buried deeply, and the dissection is forbidden. Therefore, scholars at home and abroad mainly adopt the detection of the genome DNA of the equine infectious anemia virus from peripheral blood mononuclear cells.
Disclosure of Invention
The invention aims to establish a universal and broad-spectrum equine infectious anemia fluorescence PCR detection method and a kit for the detection method.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a fluorescent PCR detection kit for equine infectious anemia virus, which contains a primer and a probe for specifically detecting equine infectious anemia virus, wherein the primer consists of an upstream primer and a downstream primer, and the sequence of the primer is as follows:
an upstream specific primer: 5 'AGACCCTRCCTGYTGAACCTGGCT 3'
A downstream specific primer: 5 'YYCCATYTTACCTGTCMYCYTGTG 3'
The probe sequence is shown as follows:
5’AAGACKTCTGTAAGTTCTCCTCTGCTG 3’
and the 5 'end of the probe is modified with a fluorescence reporter group, and the 3' end of the probe is modified with a fluorescence quenching group.
Preferably, the fluorescence reporter group is FAM, and the fluorescence quencher group is MGB.
Preferably, the kit further comprises 2 XPCRFastFire qPCR Premix, 50 XROX Reference Dye and RNase-Free ddH 2O.
Preferably, when the kit is used for horse infectious anemia virus fluorescent PCR detection, the reaction system is as follows:
2×PCRFastFire qPCR PreMix:12.5μl
upstream specific primer (10. mu.M) 0.75. mu.l
Downstream specific primer (10. mu.M) 0.75. mu.l
Probe (10. mu.M) 0.5. mu.l
Genomic DNA template 2ul
50×ROX Reference Dye:0.5μl
RNase-Free ddH2O was watered to 25. mu.l
The reaction conditions are as follows:
pre-denaturation at 98 ℃ for 1min
The fluorescence was collected at this step by denaturation at 95 ℃ for 5sec, annealing/extension at 60 ℃ for 15sec, and 40 cycles.
Furthermore, the invention also provides application of the fluorescent PCR detection kit in preparation of a reagent for detecting equine infectious anemia virus.
Preferably, the equine infectious anemia virus includes equine infectious anemia virus that is derived from both domestic and foreign sources.
Compared with the prior art, the invention has the beneficial effects that:
1. equine infectious anaemia virus members of the lentivirus family, like other retroviruses, form double-stranded viral DNA (commonly referred to as proviral DNA) in the early stages of EIAV infection by the action of viral reverse transcriptase, and the proviral DNA is further integrated into the host chromosome by the action of virally encoded integrase. Therefore, retroviruses including EIAV exist in both RNA and DNA forms in their genome, although they are RNA viruses. DNA is more stable than RNA, and the operation is more simple and convenient, so the invention adopts the fluorescence PCR method to detect the proviral DNA in the cells.
2. The primer probe combination recommended by OIE can only detect the American strain EIAV UK3 and the strain with similar sequence, and the kit can detect more strains and has the characteristic of realizing broad-spectrum detection.
3. The kit has the characteristics of high sensitivity and good specificity, detects the genomic DNA of the equine infectious anemia virus, does not need ultracentrifugation, is simple and convenient to operate, and is suitable for detecting a large number of clinical samples.
Drawings
FIG. 1 shows the amplification sequences and the positions of primers and probes according to the present invention and the methods recommended by the world animal health Organization (OIE);
FIG. 2 shows the results of primer screening;
FIG. 3 shows the efficiency of fluorescent PCR amplification using the T-tat recombinant plasmid as a template and F1/R1-3/P1_ rx primer probe combination;
FIG. 4 shows the efficiency of fluorescent PCR amplification using the T-tat recombinant plasmid as a template and F2/R2-3/P1_ rx primer probe combination;
FIG. 5 shows the specific detection results of the F1/R1-3/P1_ rx primer probe combination.
Detailed Description
The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 establishment of fluorescent PCR detection method for equine infectious anemia Virus
1. Design of primers and probes
The detection method of horse-borne poor fluorescence quantitative PCR recommended by the world animal health Organization (OIE) is designed aiming at EIAV gag gene, the sequence of the detection method is shown in Table 1, and the positions of an amplification sequence, a primer and a probe are shown in FIG. 1.
The invention analyzes and compares all (16) EIAV full-length gene sequences published in NCBI, finds a segment of relatively conserved sequence between the first exon region of tat gene and the N end of gag gene and designs degenerate primers and probes, as shown in Table 2, degenerate bases do not exceed 6bp, and the positions of amplified sequences and primers and probes are shown in figure 1.
Equine infectious anaemia virus members of the lentivirus family, like other retroviruses, form double-stranded viral DNA (commonly referred to as proviral DNA) in the early stages of EIAV infection by the action of viral reverse transcriptase, and the proviral DNA is further integrated into the host chromosome by the action of virally encoded integrase. Therefore, retroviruses including EIAV exist in both RNA and DNA forms in their genome, although they are RNA viruses. As high-speed centrifugation and fresh blood samples are needed for detecting the virus RNA, the method adopts a fluorescence PCR method to detect the genome DNA of the equine infectious anemia virus in the peripheral blood mononuclear cells in order to simplify the operation process and improve the detection sensitivity and flux.
TABLE 1 OIE recommended primer and Probe sequences
Note: r ═ A/G
TABLE 2 primer Probe sequences of the invention
Note: k is G/T; r is A/G; y is C/T; m is A/C
2. Processing of samples
1) Treatment of cell cultures: the cell culture was scraped with a cell scraper, and then tissue DNA extraction was performed using a blood/tissue/cell genome extraction kit (Tiangen).
2) Treatment of whole blood samples: to obtain Peripheral Blood Mononuclear Cells (PBMCs), whole blood was allowed to settle naturally at room temperature for 30 minutes, and the interphase between plasma and red blood cells (where the proportion of peripheral blood mononuclear cells was high) was collected. Then, tissue DNA was extracted using a blood/tissue/cell genome extraction kit (Tiangen).
3. Establishment of equine infectious anemia virus fluorescent PCR detection method
Fluorescent PCR reagent: FastFire rapid quantitative PCR reagent (Probe) (Tiangen)
Reaction system:
2×FastFire qPCR PreMix:12.5μl
upstream specific primer (10. mu.M) 0.75. mu.l
Downstream specific primer (10. mu.M) 0.75. mu.l
Probe (10. mu.M) 0.5. mu.l
Genomic DNA template 2ul
50×ROX Reference Dye:0.5μl
RNase-Free ddH2O was watered to 25. mu.l
Reaction conditions are as follows:
pre-denaturation at 98 ℃ for 1 min;
the fluorescence was collected at this step by denaturation at 95 ℃ for 5sec, annealing/extension at 60 ℃ for 15sec, and 40 cycles.
4. Screening of primers
The tissue DNA of the cell culture infected by EIAV Liaoning strain virus is extracted by using a blood/tissue/cell genome extraction kit (Tiangen), the obtained DNA sample is named as S1, different primers and probe combinations are respectively used for amplification, and the amplification result is shown in figure 2.
And (4) analyzing results: by comparison, the Ct values generated by the amplification of different primer and probe combinations on the same template (S1) are not large, wherein the Ct values obtained by the amplification of the primer and probe combination consisting of F1/R1-3, F2/R2-2 and the probe P1_ rc are slightly lower, and the amplification effect is slightly better; wherein F1/R1-3 contains a sequence of degenerate bases capable of encompassing all strains of EIAV, R2-2 is one degenerate base less than R2-3 in F2/R2-2, and the difference in Ct value is small (less than 0.5 Ct), so that primers containing degenerate bases, i.e., F1/R1-3/P1_ rx and F2/R2-3/P1_ rx, were selected for subsequent experiments.
5. Cloning of the Gene of interest
The specific DNA band was purified and recovered from the amplification product obtained from F1/R1-3(340bp-455bp, which fragment contains the sequence corresponding to 343-453bp amplified by F2/R2-3 primer) according to the Gel Extraction Kit DNA recovery Kit. And the DNA fragment is connected to a pMD18T vector, DH5 alpha is transformed, a positive clone is selected and named as T-tat, and the T-tat is sent to Jilin Ku Mei biological company for sequencing and sequence analysis.
As a result: the T-tat recombinant plasmid was sequenced by the universal primer. Sequencing analysis shows that the cloned fragment is compared with the known equine infectious anemia virus sequence, and the result shows that the homology of the sequence is 100 percent, which indicates that the cloned sequence is correct.
6. Efficiency of amplification
T-tat recombinant plasmid is used as a template, 10-fold gradient dilution is carried out, F1/R1-3/P1_ rx primer probe combination is used for carrying out amplification of fluorescence PCR, each dilution is repeated for 2 times, the amplification efficiency is 96.7%, and the result is shown in figure 3.
T-tat recombinant plasmid is used as a template, 10-fold gradient dilution is carried out, F2/R2-3/P1_ rx primer probe combination is used for carrying out amplification of fluorescence PCR, each dilution is repeated for 2 times, the amplification efficiency is 94.4%, and the result is shown in figure 4.
As a result: the amplification efficiency of the F1/R1-3/P1_ rx primer probe combination is higher than that of the F2/R2-3/P1_ rx primer probe combination, so the screened primer probe combination is F1/R1-3/P1_ rx.
7. Sensitivity detection
Taking T-tat recombinant plasmids as templates, and carrying out 10-fold gradient dilution, wherein the template concentrations are respectively 105、104、103、102、101、100Copy/. mu.l; or diluting the extracted EIAV virus DNA by 10 times, and performing fluorescent PCR amplification by using a primer probe combination F1/R1-3/P1_ rx. With H2O as a negative control.
As a result: the sensitivity of the primer probe combination F1/R1-3/P1_ rx is 10 copies/mu l of template and 145pg/ul of tissue DNA in a cell sample.
8. Specificity detection
Nucleic acids of EIAV virus, equine influenza virus, equine arteritis virus, equine herpes type I virus, equine herpes type IV virus, equine abortion salmonella, and equine streptococcus respectively serve as templates, and fluorescence PCR amplification is carried out by using a primer probe combination F1/R1-3/P1_ rx.
As a result: this method produces a specific amplification reaction only for EIAV virus, but does not amplify other equine infectious disease viruses or bacteria, and the results are shown in FIG. 5.
9. Verification of EIAV universal nucleic acid detection
As only the Chinese strains (liaoning strains, Vaccine strains, DLV2-6 and the like) and the UK3 strains with EIAV are stored in the laboratory, under the condition that foreign strains cannot be obtained, sequences on different strains at corresponding positions of target fragments obtained by the method and the OIE amplification method are synthesized, the two synthesized target fragment sequences are connected through 10T (the amplified fragments of the method + 10T + OIE amplified fragments), then specific gene sequences of representative strains are inserted into a pMV vector, and the obtained synthetic gene plasmids are used as templates to carry out fluorescence PCR amplification respectively. The mass of the synthetic gene positive plasmid is about 3-5ug, 1ml of DEPC water is respectively added, and the mixture is diluted by 100 times to be used as a template of fluorescence PCR.
To obtain equine infectious oligovirus genomic DNA in cell cultures, cell cultures infected with EIAV lioning, Vaccine, DLV2-6, and UK3 strains were scraped with a cell scraper, and then tissue DNA was extracted using a blood/tissue/cell genome extraction kit (tiangen).
The primer probe combination and the primer probe combination recommended by OIE are used for carrying out fluorescence PCR detection on strains from different sources, and the results are shown in the following table 3.
Table 3 results of detection of strains from different sources using the primer probe combination of the present invention and OIE primer probe combination
As can be seen from Table 3, only the American strain EIAVUK3 and the strain with similar sequence can be detected by using the primer probe combination recommended by OIE, and more strains can be detected by using the primer probe combination F1/R1-3/P1_ rx provided by the invention, so that the primer probe combination has the characteristic of being capable of realizing broad-spectrum detection.
10. Detection of clinical blood samples
For fresh whole blood samples, the whole blood was allowed to settle naturally at room temperature for 30 minutes, and the interphase between plasma and red blood cells (i.e., peripheral blood mononuclear cells) was collected. Then, tissue DNA was extracted using a blood/tissue/cell genome extraction kit (Tiangen).
For whole blood and serum that have been frozen and thawed, extraction of tissue DNA can be performed directly using a blood/tissue/cell genome extraction kit (tiangen).
As China eliminates the horse poor transmission, and no related clinical cases exist in recent years, the research adopts positive samples reserved in laboratories to carry out detection (such as freeze-thaw whole blood and horse poor transmission positive serum) and collects clinical samples (fresh whole blood and serum).
The results of fluorescence PCR detection of the above samples using the primer probe combination of the present invention are shown in Table 4.
TABLE 4 detection of clinical samples Using primer-probe combinations of the invention
| Sample numbering | Sample type | Fluorescent PCR (Ct value) | Remarks for |
| 1 | Fresh whole blood | no Ct | Clinical samples |
| 2 | Fresh foodBlood circulation | no Ct | Clinical samples |
| 3 | Fresh whole blood | no Ct | |
| 4 | Fresh whole blood | no Ct | Clinical samples |
| 5 | Fresh whole blood | no Ct | Clinical samples |
| 6 | Fresh whole blood | no Ct | Clinical samples |
| 7 | Fresh whole blood | no Ct | |
| 8 | Freeze thawing of whole blood | 30.53 | Testing infected animals |
| 9 | Freeze thawing of whole blood | 26.24 | Testing |
| 10 | Poor positive serum of horse passing poverty | 29.41 | Laboratory retained samples |
| 11 | Serum | no Ct | |
| 12 | Serum | no Ct | Clinical samples |
| 13 | Serum | no Ct | |
| 14 | Serum | no Ct | Clinical samples |
| 15 | Serum | no Ct | Clinical samples |
As can be seen from Table 4, the primer probe combination of the present invention can detect equine poor genomic DNA in clinical samples, and the detection results are negative for other non-equine poor clinical cases.
Example 2 Assembly and detection method of fluorescent PCR detection kit for equine infectious anemia virus
Assembling the kit:
1. primers and probes
The primer is used for specifically detecting the equine infectious anemia virus, the primer consists of an upstream primer and a downstream primer, and the sequence of the primer is as follows:
an upstream specific primer: 5 'AGACCCTRCCTGYTGAACCTGGCT 3'
A downstream specific primer: 5 'YYCCATYTTACCTGTCMYCYTGTG 3'
The probe sequence is shown as follows:
FAM-AAGACKTCTGTAAGTTCTCCTCTGCTG-MGB
2、2×PCRFastFire qPCR PreMix
3、50×ROX Reference Dye
4、RNase-Free ddH2O
the detection method comprises the following steps:
when the kit is used for PCR fluorescent PCR detection of the equine infectious anemia virus, the reaction system is as follows:
2×FastFire qPCR PreMixPCR:12.5μl
upstream specific primer (10. mu.M) 0.75. mu.l
Downstream specific primer (10. mu.M) 0.75. mu.l
Probe (10. mu.M) 0.5. mu.l
Genomic DNA template 2ul
50×ROX Reference Dye:0.5μl
RNase-Free ddH2O was watered to 25. mu.l
The reaction conditions are as follows:
pre-denaturation at 98 ℃ for 1min
The fluorescence was collected at this step by denaturation at 95 ℃ for 5sec, annealing/extension at 60 ℃ for 15sec, and 40 cycles.
Claims (6)
1. The Equine Infectious Anemia Virus (EIAV) fluorescent PCR detection kit is characterized in that the fluorescent PCR detection kit contains a primer and a probe for specifically detecting the Equine infectious anemia virus, wherein the primer consists of an upstream primer and a downstream primer, and the sequence of the primer is as follows:
an upstream specific primer: 5 'AGACCCTRCCTGYTGAACCTGGCT 3'
A downstream specific primer: 5 'YYCCATYTTACCTGTCMYCYTGTG 3'
The probe sequence is shown as follows:
5’AAGACKTCTGTAAGTTCTCCTCTGCTG 3’
and the 5 'end of the probe is modified with a fluorescence reporter group, and the 3' end of the probe is modified with a fluorescence quenching group.
2. The fluorescence PCR detection kit of claim 1, wherein the fluorescence reporter is FAM and the fluorescence quencher is MGB.
3. The fluorescence PCR assay kit of claim 1, wherein said kit further comprises 2 XPCR FastFire qPCRPreMix, 50 XPOX Reference Dye, and RNase-FreedH 2O.
4. The fluorescence PCR detection kit of claim 1, wherein when the kit is used for PCR fluorescence PCR detection of equine infectious anemia virus, the reaction system is as follows:
2×FastFire qPCRPreMix:12.5μl
upstream specific primer (10. mu.M) 0.75. mu.l
Downstream specific primer (10. mu.M) 0.75. mu.l
Probe (10. mu.M) 0.5. mu.l
Genomic DNA template 2ul
50×ROXReference Dye:0.5μl
RNase-Free ddH2O was watered to 25. mu.l
The reaction conditions are as follows:
pre-denaturation at 98 ℃ for 1min
The fluorescence was collected at this step by denaturation at 95 ℃ for 5sec, annealing/extension at 60 ℃ for 15sec, and 40 cycles.
5. Use of the fluorescent PCR assay kit of any one of claims 1 to 4 in the preparation of a reagent for the detection of equine infectious anemia virus.
6. The use of claim 5, wherein said equine infectious anemia virus comprises equine infectious anemia virus of both domestic and foreign origin.
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| 耿庆华等: "马传染性贫血病毒阿根廷流行毒株前病毒gag和gp90基因在马体内的变异分析", 《畜牧兽医学报》 * |
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