Primer set and kit for detecting Zika virus (ZIKV) based on one-step fluorescent quantitative nested RT-PCR

A RT-PCR, fluorescent quantitative technology, applied in the biological field, can solve the problems of detection sensitivity to be improved, cumbersome steps, sample contamination, etc., and achieve high sensitivity, good repeatability, and good primer specificity

Pending Publication Date: 2018-10-02
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The inventor’s previous patent (CN 105861753) disclosed a nested RT-PCR method, primers and kits for detecting Zika virus. Nested RT-PCR needs to b

Method used

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  • Primer set and kit for detecting Zika virus (ZIKV) based on one-step fluorescent quantitative nested RT-PCR
  • Primer set and kit for detecting Zika virus (ZIKV) based on one-step fluorescent quantitative nested RT-PCR
  • Primer set and kit for detecting Zika virus (ZIKV) based on one-step fluorescent quantitative nested RT-PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The design and synthesis of embodiment 1 primer

[0039] Using all existing ZIKV full-length genome sequences in GenBank for multiple sequence alignment, select the conserved region (575bp, SEQ ID No: 5) of the ZIKV envelope protein (envelope protein) to design primers (Table 1). Synthetic;

[0040] Table 1 Specific primers for one-step fluorescent quantitative nested RT-PCR detection

[0041]

[0042] M in the primer sequence stands for A or C, R stands for A or G, Y stands for C or T, S stands for C or G

Embodiment 2

[0043] Example 2 Viral RNA extraction and one-step fluorescent quantitative nested RT-PCR

[0044] The viral RNA extraction process was carried out according to the instructions of the QIAamp Viral RNA Mini Kit. One-step fluorescent quantitative nested RT-PCR was carried out with the primer set in Example 1, and the reaction system was as follows:

[0045]

[0046] The reaction conditions are: reverse transcription at 42°C for 10 minutes, pre-denaturation at 95°C for 2 minutes, outer PCR (95°C for 15 sec, 65°C for 15 sec, 72°C for 35 sec) x 15 cycles, inner PCR (95°C for 15 sec, 48°C for 15 sec, 72°C 30sec)×40 cycles, melting curve analysis.

Embodiment 3

[0047] Embodiment 3 one-step fluorescent quantitative nested RT-PCR specificity test

[0048] Select 4 strains of ZIKV (all Asian strains), 1 strain of ZIKV MR_766 (African strain), 4 strains of dengue virus (1 strain for each type 1-4), 1 strain of flavivirus and 1 strain of influenza A virus for H5N6 cell culture After clearing, extract RNA for one-step fluorescence quantitative nested RT-PCR specificity test, each take 1 μL of total RNA as a template for reaction, and nuclease-free dH 2 O is used as negative control; Reaction system and reaction condition are identical with above-mentioned embodiment 2.

[0049] The result is as figure 1 As shown, 5 strains of ZIKV can be amplified specifically, but 4 strains of dengue virus, flavivirus and influenza A virus H5N6 will not be amplified, indicating that the one-step fluorescent quantitative method of ZIKV involved in the present invention Formula RT-PCR primers have good amplification specificity.

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Abstract

The invention relates to a primer set and kit for detecting ZIKV based on one-step fluorescent quantitative nested RT-PCR, belonging to the field of biotechnology. The primer set comprises a pair of external primers and a pair of internal primers, wherein the upstream and downstream sequences of the external primers are as shown in SEQ ID No. 1-2, and the upstream and downstream sequences of the internal primers are as shown in SEQ ID No. 3-4. The one-step fluorescent quantitative nested RT-PCR primers for ZIKV designed in the invention have good specificity, high sensitivity and good repeatability. A designed one-step fluorescence quantitative nested RT-PCR detection method can sensitively, accurately, stably and rapidly detect the presence of ZIKV and is capable of detecting viruses withthe lowest concentration of 6.4*10<-1> TCID50/mL. The one-step fluorescence quantitative nested RT-PCR detection method provides a technical means for rapid and sensitive detection of ZIKV, and is ofgreat significance to the rapid clinical diagnosis and public prevention and control of ZIKV at present.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a primer set and a kit for detecting Zika virus based on one-step fluorescent quantitative nested RT-PCR. Background technique [0002] Zika virus (zika virus, ZIKV) was first identified in 1947 in a rhesus monkey sample in the Zika jungle of Uganda through the Yellow Fever Surveillance Network, and its name is also derived from this, and was reported in Uganda and Tanzania in 1952 Can infect humans. ZIKV belongs to the Flavivirus genus of the family Flaviviridae in classification, and is a single-stranded positive-sense RNA virus with a genome length of about 10.8 kb. ZIKV particles are spherical, with a diameter of about 40-70nm. The ZIKV genome encodes a single open reading frame (open reading frame), which encodes a polyprotein, which is cleaved into different functional proteins after translation, including 3 structural proteins (C, prM / M, E)) and 7 Nonstructu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12R1/93
CPCC12Q1/6848C12Q1/701C12Q2549/119C12Q2521/107C12Q2563/107C12Q2545/114
Inventor 高福刘映霞毕玉海王强王颂基杨扬郑海霞李善琴
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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