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Multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and detection method of multiplex RT-PCR kit

A technology of BTV-20 and BTV-11 is applied in the field of molecular biology detection methods and detection reagents, which can solve the problems of high cost, long time, and inability to type viruses, and achieves reduction of pollution, avoidance of chemical pollution, and reduction of primers. cost effect

Active Publication Date: 2018-07-31
重庆海关技术中心
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  • Summary
  • 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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  • Multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and detection method of multiplex RT-PCR kit
  • Multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and detection method of multiplex RT-PCR kit
  • Multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and detection method of multiplex RT-PCR kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, design and synthesis of primers

[0047] According to the VP2 gene sequences of BTV11, 17, 20, 23, and 24 viruses published on the Genbank website, the specific primers for 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:

[0048] BTV-11-specific upstream primer SEQ ID NO.1: 5'-CGGTTGCGAATAACTCAT-3';

[0049] BTV-11-specific downstream primer SEQ ID NO.2: 5'-ATTGTATACGCATGTCGA-3';

[0050] BTV-17 specific upstream primer SEQ ID NO.3: 5'-GAGTTCGTCATTCCAGTC-3';

[0051] BTV-17-specific downstrea...

Embodiment 2

[0080] Embodiment 2, positive recombinant plasmid construction

[0081] Viral RNA was extracted according to the TaKaRa Mini BEST Viral RNA / DNA Extraction Kit. Use each specific primer separately 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

[0082] Embodiment 3 single-plex RT-PCR construction and optimization

[0083] 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.

[0084] 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 siz...

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Abstract

The invention discloses a multiplex RT-PCR kit for BTV-11, BTV-17, BTV-20, BTV-23 and BTV-24 genotype identification and a detection method of the multiplex RT-PCR kit. The multiplex RT-PCR kit is used for simultaneously discriminating and detecting bluetongue virus types 11, 17, 20, 23 and 24 with a single tube. According to the method, 5 pairs of PCR specific primers are designed according to conserved regions of VP2 gene sequences of various types of viruses, in addition, a pair of non-biogenic-derivation universal primers are synthesized with reference to a GeXP principle, the universal primers are separately added to upstream and downstream of each pair of specific primers to form 5 pair of specific embedded primers, reverse transcription is carried out with the specific primers, andfinally, multiplex PCR is constructed by adopting the universal primers and the specific embedded primers. By utilizing an optimized multiplex PCR system and conditions, one or more of the 5 genotypesof the bluetongue viruses can be simultaneously discriminated and detected with the single tube, specific amplification to other types of BTV, PPRV and FMDV nucleic acid is avoided, and the minimum detection concentration can reach a pg level. The method disclosed by the invention is high in sensitivity, high in specificity and easy in result observation and is timesaving and laborsaving.

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 11, 17, 20, 23, and 24 genotype multiplex RT-PCR detection kit and detection 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 perfo...

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 聂福平杨俊王国民王昱黄秋华李贤良
Owner 重庆海关技术中心
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