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Multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and detection method of BTV genotypes 2, 3, 4, 7 and 12

A BTV-12, RT-PCR technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of time-consuming, high cost, inability to type viruses, etc., to avoid chemical pollution and reduce Contamination, effect of reducing primer cost

Active Publication Date: 2018-08-10
重庆海关技术中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Viruses cannot be typed except for VNT. Although VNT can be typed, it takes a long time, the cost is too high, and the quality of serum used for detection is high, so it is not suitable for routine detection.
Scholars at home and abroad have done related research on virus typing, but they can only type one type of virus with a single tube, and the method of typing multiple types of viruses with a single tube with high throughput has not been reported yet

Method used

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  • Multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and detection method of BTV genotypes 2, 3, 4, 7 and 12
  • Multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and detection method of BTV genotypes 2, 3, 4, 7 and 12
  • Multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and detection method of BTV genotypes 2, 3, 4, 7 and 12

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, design and synthesis of primers

[0046] According to the VP2 gene sequences of BTV-2, 3, 4, 7, and 12 viruses published on the Genbank website, primers specific to these 5 types of viruses were designed using Primer Premier 5.0 biological software, which were respectively marked as SEQ ID NO. 1-SEQ ID NO.10; refer to Gexp-PCR universal primers, marked as SEQ ID NO.11, SEQ ID NO.12; respectively add the upstream and downstream of the universal primers to the 5' end of each specific upstream and downstream primers to form a specific chimera Primers, marked as SEQ ID NO.13-SEQ ID NO.22, all primers were sent to BGI for synthesis. The specific primer sequences are as follows:

[0047] BTV-2 specific upstream primer SEQ ID NO.1: 5'-TACTGAGGTTGAAGAGAATCC-3'

[0048] BTV-2 specific downstream primer SEQ ID NO.2: 5'-ATCAAGCGTGCGAATGTT-3'

[0049] BTV-3 specific upstream primer SEQ ID NO.3: 5'-TATCCATCAGGCTCTCGTA-3'

[0050] BTV-3 specific downstream primer SE...

Embodiment 2

[0079] Embodiment 2, positive recombinant plasmid construction

[0080] Viral RNA was extracted according to the TaKaRa Mini BEST Viral RNA / DNA Extraction Kit. Use each specific primer according to PrimeScript TM One Step RT-PCR kit amplification; recover the target fragment according to the OMEGA GelEetraction Kit instructions, connect it to the PMD19-T vector and transform it into DH5a, after enrichment, extract the plasmid according to the kit AXGYEN AxyPreP Plasmid Minippep kit, and perform PCR For identification, the PCR-positive plasmid is sent to BGI for sequencing, and the sequencing results are compared in NCBI. If the comparison is correct, it is a positive plasmid. Use a spectrophotometer to measure the concentration of the positive recombinant plasmid and calculate the copy number.

Embodiment 3

[0081] Embodiment 3 single-plex RT-PCR construction and optimization

[0082]Construct a 25 μL single-plex PCR detection system using the above positive plasmid as a template: Permix Taq 12.5 μL, general F and R (25 μmol / L) each 1 μL, specific chimeric F and R (10 μmol / L) each 0.25 μL, template 1 μL, Make up to 25 μL with sterilized water. Reaction program: 94°C for 5min; 94°C for 30s, 58°C for 30s, 72°C for 30s, 10 cycles; 95°C for 30s, 50°C for 30s, 72°C for 30s, 25 cycles; 72°C for 5min, 4°C for storage. The product was analyzed by capillary electrophoresis. On this basis, the annealing temperature of specific chimeric primers (55°C, 56°C, 57°C, 58°C, 59°C, 60°C) was optimized, and the concentration of specific chimeric primers (0.05 μmol / L, 0.1 μmol / L, 0.15μmol / L, 0.20μmol / L, 0.25μmol / L) optimization.

[0083] refer to figure 1 The results showed that each serotype was amplified with a product consistent with the size of the target product. The theoretical product si...

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Abstract

The invention discloses a multi-RT (reverse transcription)-PCR (polymerase chain reaction) reagent kit for genotyping identification of BTV (bluetongue virus) genotypes 2, 3, 4, 7 and 12 and a detection method of the BTV genotypes 2, 3, 4, 7 and 12, which are used for simultaneous identification and detection of the BTV genotypes 2, 3, 4, 7 and 12 through a single tube. According to different genotype virus VP2 gene sequence conservation areas of bluetongue, 5 pairs of PCR specific primers are designed in the method, a pair of non-biologically derived universal primers are synthesized by utilizing a GeXP principle, the universal primers are respectively added to the upstream and downstream of each pair of specific primers to form 5 pairs of specific chimeric primers, the specific primers are used for performing inverse transcription, and multi-PCR is constructed by combining the universal primers with the specific chimeric primers. By utilizing an optimized multi-PCR system and condition, one or more types of 5 genotypes, i.e., BTV genotypes 2, 3, 4, 7 and 12 can be simultaneously identified and detected through the single tube, other types of PTV, PPRV (peste des petits ruminantsvirus) and FMDV (foot-and-mouth disease virus) nucleic acids are not subjected to specific amplification, and the lowest detection concentration can reach a pg level. According to the method constructed by the invention, the sensitivity is high, the specificity is strong, the time and labor are saved, and the result is easy to observe.

Description

technical field [0001] The invention belongs to the field of molecular biology detection methods and detection reagents, and the technical field of PCR. Specifically, it relates to a bluetongue virus (BTV) type 2, type 3, type 4, type 7, and type 12 multiplex RT-PCR detection kit and method. Background technique [0002] Bluetongue (Bluetongue, BT) is caused by Bluetongue virus (BTV), a member of the Reoviridae Orbiviridae genus, and is a non-contact infectious disease of ruminants with insects as the medium. Widely distributed, bluetongue has been reported on every continent except Antarctica. BTV can infect most ruminants. Sheep are the most susceptible and show typical clinical symptoms. Cattle and goats are mostly recessively infected and have no obvious symptoms, but they can carry the virus for a long time. Wild animals and camels can also be infected with the disease. Due to the disease and death of sick sheep, or even if they are not dead, their production performa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2545/113
Inventor 聂福平王昱黄秋华王国民杨俊李贤良
Owner 重庆海关技术中心
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