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Zika virus real-time fluorescence quantitative RT-RPA detection primers, probes and detection kit

A real-time fluorescence quantitative and Zika virus technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of high price, complicated operation, large volume, etc., and achieve fast detection speed , good effect of specificity

Active Publication Date: 2020-01-17
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0003] Molecular amplification is an important part of nucleic acid molecular detection. PCR technology relies on temperature changes to achieve amplification reactions such as denaturation, renaturation, and extension. The completion of amplification technology depends on sophisticated and complex large-scale instruments, such as temperature-controlled adjustment instruments and complex These equipment are bulky, expensive and complicated to operate, and are not suitable for on-site emergency detection tasks during outbreaks

Method used

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  • Zika virus real-time fluorescence quantitative RT-RPA detection primers, probes and detection kit
  • Zika virus real-time fluorescence quantitative RT-RPA detection primers, probes and detection kit
  • Zika virus real-time fluorescence quantitative RT-RPA detection primers, probes and detection kit

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Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Establishment of real-time fluorescent quantitative RT-RPA detection method for Zika virus

[0045] 1. Preparation of in vitro transcribed RNA

[0046] The PCR method is used to introduce the T7 promoter into the upstream of the positive-strand virus reading frame and the downstream of the negative-strand virus reading frame, so as to obtain RNA consistent with the viral genome sequence after in vitro transcription. Use the cDNA synthesized by reverse transcription of viral RNA or the plasmid containing the artificially synthesized target gene as a template, and use the FastStart High Fidelity PCR system to prepare the following system:

[0047]

[0048]

[0049] The sequences of forward and reverse primers are as follows:

[0050] Forward primer: 5′-TAATACGACTCACTATAGGATGTGGGGTGCTCGG-3′

[0051] Reverse primer: 5′-TGCAGTCACCACTGACCTTACTAAGTT-3′

[0052] The full-length ZIKA NS1 gene was amplified, and its sequence is shown in SEQ ID NO:4.

[0053] T...

Embodiment 2

[0120] The optimization of embodiment 2 RPA primer, probe

[0121] 1. Primer / probe design

[0122] Through comparison and analysis of primer-probe design software, according to the design principles, the conserved region of the ZIKV NS1 fragment was selected to design 5 upstream and downstream primers and 3 probes. The sequences are shown in Table 3.

[0123] Table 3 ZIKV NS1 exo-RT amplification primer sequences

[0124]

[0125]

[0126] 2. Probe screening

[0127] By combining 5 upstream primers and 1 downstream primer one by one, the combinations are shown in Table 4, and the 3 probes were screened respectively. according to Figure 5 The results showed that the five upstream and downstream primer combinations all had better amplification reactions with the probe ZIKV-EXO-P1-4 (2662-2706), and the required reaction time was shorter, so the probe ZIKV-EXO-P1- 4 is better than ZIKV-EXO-P1-3 and ZIKV-EXO-P1-6.

[0128] Table 4 Screening of probes by 5 kinds of prim...

Embodiment 3

[0152] The optimization of embodiment 3 reaction system

[0153] 1. Optimization of magnesium acetate content in the real-time fluorescence RT-RPA detection system

[0154] Magnesium acetate is the key to start the whole RT-RPA reaction, and different contents of magnesium acetate have different effects on the reaction time. With reference to relevant literature, magnesium acetate reaction condition is preferably between 12mM-20mM, therefore, the first round of screening is that final concentration is 280mM magnesium acetate respectively with final concentration being 11.76mM (2.1 μ l), 13.44mM (2.4 μ l), 15.12 Six gradients of mM (2.7 μl), 16.8 mM (3.0 μl), 18.48 mM (3.3 μl), and 20.16 mM (3.6 μl) were used for the experiment and repeated twice.

[0155] Figure 10 Middle (A and B) shows that the time required for the reaction between 15.12mM (2.7μl)-20.16mM (3.6μl) is the least, and the optimal reaction concentration may be located in this interval. In the second round of...

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Abstract

The invention provides Zika virus real-time fluorescence quantitative RT-RPA detection primers, probes and a detection kit. The kit comprises the primers and the probes for detecting Zika virus basedon a RPA technology, and upstream and downstream primers and probe sequences are respectively shown in SEQ ID NO. 1-3. The real-time fluorescence quantitative RT-RPA technology is adopted for the first time to establish a method for rapidly detecting the Zika virus, and through specificity, sensitivity and stability evaluation, the primers, the probes and the detection kit can be used for clinicalon-site detection, and provides a sensitive and reliable new method for on-site detection of the Zika virus.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to Zika virus real-time fluorescence quantitative RT-RPA detection primers and probes and a detection kit. Background technique [0002] The recent increase in the number of patients with congenital microcephaly and other neurological disorders that may be associated with Zika virus infection has led to an increased need to detect and diagnose Zika virus infection. Viral nucleic acid detection technology can specifically detect virus target genes, is more sensitive and fast, and greatly avoids the biosafety risk of using infectious viruses. It has unique advantages in the detection and diagnosis of early Zika virus infection, and is suitable for Zika virus Detection of specimens in the acute phase of viral diseases. [0003] Molecular amplification is an important part of nucleic acid molecular detection. PCR technology relies on temperature changes to achieve amplif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2531/119C12Q2521/107C12Q2521/507C12Q2537/1376C12Q2522/101C12Q2527/127
Inventor 李阿茜梁米芳李建东李德新刘洋芜为李川
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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