One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof

A Zika virus and reverse transcription technology, applied in the field of biological sciences, can solve problems such as the inability to effectively reduce false positive results and the feasibility of detection results needs to be improved, and achieve the effect of high accuracy

Inactive Publication Date: 2016-09-14
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical solution of this patent application: 1) can only detect the Asian type of Zika virus; 2) it uses the fluorescent reverse transcription PCR of the hydrolysis probe (fluorescent oligonucleotide probe) for detection, so when performing the detection of related samples There are only amplification curves but no m

Method used

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  • One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof
  • One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof
  • One step method inverse transcription PCR kit for detecting and differentiating Zika viruses and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] This example 1 provides a one-step reverse transcription FRET-PCR kit for detecting and typing two genotypes of Zika virus, including primers and probes, as well as PCR buffer and hot start Taq enzyme , dNTP, FRET-PCR standard, and double distilled water PCR negative control;

[0055] Wherein, the primers are upstream primers and downstream primers; the probes are 6-FAM probes and LCRed640 probes; the nucleotide sequences of the primers and probes are respectively as follows:

[0056] Upstream primer: 5'-AGCTATTATGCCGCCACCATCC-3' (SEQ ID No.1);

[0057] Downstream primer: 5'-CAGAGTGTCACACGGCTCAGCC-3' (SEQ ID No.2)

[0058] 6-FAM probe: 5'-CCATGCTGGTGCAAAGCTATGG-FAM-3' (SEQ ID No.3)

[0059] LCRed640 probe: 5'-LCRed640-TGGAACATAGTTCGTCTCAAAGAGTGG-phosphate group-3 (SEQ ID No.4)

[0060] Among them, the 6-FAM probe is a donor probe, which is labeled with the donor dye FAM at its 3' end; the LCRed640 probe is an acceptor probe, and is labeled with the acceptor dye LCRed...

Embodiment 2

[0084] In Example 2, the sensitivity, specificity and amplification efficiency of the one-step reverse transcription FRET-PCR detection method of the present invention were verified by plasmid standards carrying the African type of Zika virus and the Asian type of Zika virus.

[0085] 1) Determination of the sensitivity of the detection method of the present invention

[0086] Step A: Preparation of DNA plasmid standards

[0087] GenScript (GenScript Biotechnology, Nanjing, China) synthesized the DNA sequences of the target fragments of Zika virus 2 genotypes (ZIKV-Asian, ZIKV-African). Based on the molecular weight and absolute weight of the composition, the absolute number of gene copies contained in the composition is calculated. Subsequently, the composition was diluted to prepare dilution reagents per reaction containing 1 x 10 4 copy, 1×10 3 copy, 1×10 2 copy, 1×10 1 copy, 1×10 0 A copy of the target gene was used as a standard. Use the system of the present inven...

Embodiment 3

[0113] In Example 3, the target nucleic acid fragment was synthesized by gene and the introduced plasmid was transcribed and synthesized viral RNA nucleic acid in vitro by a commercial kit, and then the one-step reverse transcription FRET-PCR detection method in the present invention was verified. In Example 3, the Taking the detection of the Asian type of Zika virus as an example, the specific operation process is as follows:

[0114] Step A: GenScript (GenScript Biotechnology, Nanjing, China) synthesized the plasmid pUC57 containing the Zika virus Asian type target fragment DNA (SEQ ID No.5) and T7 promoter, and used appropriate restriction endonucleases Enzyme (Sac I, Treasure Bio, Dalian) was used to digest the plasmid; PCR amplification was used to increase the concentration of the target DNA; a commercial transcription kit ( Kit, by life USA)) transcribe the PCR amplified product and obtain the RNA product; after the transcription is completed, the transcribed produ...

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Abstract

The invention provides a primer, probe, and kit for detecting and differentiating Zika viruses through one step method inverse transcription PCR, a detection method and applications thereof. The PCR kit can be sensitively, specifically, efficiently, and rapidly detect and differentiate two genotypes of Zika viruses namely Asia type and Africa type at the same time. The principle of the kit is that on the basis of an inverse transcription PCR technology, a one step method FRET-PCR system, which can simultaneously detect and differentiate two Zika viruses, is established. The system is built on the basis of two genotypes of Zika viruses and whole gene sequences of other pathogens having a high homology with two genotypes of Zika viruses; a relatively conservative section is selected to design the primer and probe, through specific amplification, positive samples can be detected and screened from clinical samples sensitively and rapidly; then according to the difference of melting temperature (TM) values in a high resolution melting curve, the Asia type and Africa type of Zika viruses can be differentiated credibly; operation of the kit and the detection method is convenient and efficient, and the kit and method are suitable for detecting a large amount of samples.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to a kit for one-step reverse transcription PCR detection and typing of Zika virus and a detection method thereof. Background technique [0002] Zika virus disease, also known as Zika fever (Zika fever), is caused by Zika virus (Zika virus, ZIKAV) and passes through Aedes mosquitoes (mainly including Aedes aegypti, Aedes albopictus, Aedes africanus) Africana) and other acute viral vector-borne diseases transmitted by blood-sucking insects. The disease can also be transmitted from a pregnant woman to the fetus or infant during pregnancy or delivery, or through sexual transmission or blood transfusion. The incubation period of Zika virus infection is about 3-12 days. The main clinical symptoms are fever, rash, severe joint pain, muscle pain, and conjunctivitis. In cases of pediatric infection, changes in the nervous system, eyes, and hearing may also occur. Infection in p...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/686C12Q2565/101C12Q2545/113C12Q2527/107C12Q2563/107
Inventor 王成明张继垒王瑶瑶有金凤仇海香黄可
Owner YANGZHOU UNIV
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