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Nucleic acid set, kit and detection method for detecting Hantaan viruses through RPA (recombinase polymerase amplification)

A technology of Hantaan virus and a kit, applied in the biological field, can solve the problems of rapid detection of Hantaan virus, inability to separate from price automatic instruments, failure to perform typing, etc., and achieves short detection time, short response time, and simplicity. carry effect

Active Publication Date: 2019-10-25
中国人民解放军东部战区疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technical solution cannot be classified and cannot be separated from expensive automatic instruments, so it is difficult to apply to the current situation of on-site detection and other scenarios
[0012] At present, there is no method for rapid detection of Hantaan virus using recombinase polymerase constant temperature amplification technology (RPA technology)

Method used

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  • Nucleic acid set, kit and detection method for detecting Hantaan viruses through RPA (recombinase polymerase amplification)
  • Nucleic acid set, kit and detection method for detecting Hantaan viruses through RPA (recombinase polymerase amplification)
  • Nucleic acid set, kit and detection method for detecting Hantaan viruses through RPA (recombinase polymerase amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, design and screening of Hantaan virus HTNV primers and probes

[0054] (1) Design of primers and probes

[0055] The inventor's laboratory has preserved the positive plasmid standard product (GeneBank: M14626.1) of the S gene of the conservative segment of the Hantaan virus HTNV standard strain 76-118, i.e. the nucleotide sequence shown in SEQ ID NO.1. S gene recombinant plasmid using in vitro transcription kit After Systems-T7 (Promega, the U.S.) was transcribed into RNA in vitro, the concentration was measured using NanoDrop, according to the formula (6.02 × 10 23 copy number / mole)×(concentration ng / μL×10 -9 ) / (RNA length×340)=copies / μL to convert copy number, take 10 5 Copies / μL RNA 1 μL using PrimeScript TM The 1st StrandcDNA Synthesis Kit (Takara, Japan) was used for reverse transcription to obtain cDNA, which was used as a template in the subsequent screening of primers and probes and optimization of the reaction system in Example 1 and Exampl...

Embodiment 2

[0067] Embodiment 2: Optimization of RPA reaction system, amplification and detection conditions

[0068] In the process of primer screening, the disposable nucleic acid detection device is still unstable, so the RPA reaction system, amplification and detection conditions need to be optimized

[0069] (1) Primer probe concentration

[0070] The concentration of the forward primer was kept constant at 10 μM, the concentration of the probe was set to 10 μM, 5 μM, and 2.5 μM, and the concentration gradient of the reverse primer was 10 μM, 5 μM, and 2.5 μM. The reverse primers were combined into 9 groups, the combination numbers are shown in Table 3, and a negative control was set for each group. The 50 μL RPA reaction system and amplification conditions were the same as in Example 1, and the results were interpreted using a disposable nucleic acid detection device. Table 3 Combination numbers of reverse primer concentration and probe concentration.

[0071] Table 3. Probe Concen...

Embodiment 3

[0080] Embodiment 3: Sensitivity evaluation of RPA detection

[0081] With the Hantaan virus HTNV recombinant plasmid mentioned in Example 1, first use the in vitro transcription kit After Systems-T7 (Promega, the U.S.) was transcribed into RNA in vitro, the concentration was measured using NanoDrop, according to the formula (6.02 × 10 23 copy number / mole)×(concentration ng / μL×10 -9 ) / (RNA length×340)=copies / μL conversion copy number, ten-fold dilution to get 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Copies / μL, 10copies / μL, 1copies / μL of RNA, take 1μL each and use PrimeScript TM The 1st Strand cDNASynthesis Kit (Takara, Japan) was reverse-transcribed to obtain gradient cDNA, each using 1 μL of cDNA as a template to add the optimal reaction system determined in Example 2, and using the optimal primer combination screened in Example 1 for RPA detection. After mixing, amplify at 37°C for 17.5 minutes, take out the reaction tube at the 4th minut...

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Abstract

The invention relates to a nucleic acid set, a kit and a detection method for detecting Hantaan viruses through RPA (recombinase polymerase amplification). The nucleic acid set comprises a primer pairand a probe. The primer pair comprises a forward primer and a reverse primer, the sequence of the forward primer is SEQ ID NO. 2, the sequence of the reverse primer is SEQ ID NO. 3, and the sequenceof the probe is SEQ ID NO. 4. The kit comprises the nucleic acid set, and is applied to the detection method. Amplification can be realized at a temperature near to the body temperature, and visual discrimination of amplified products can be realized by a disposable nucleic acid detecting device. The nucleic acid set, the kit and the detection method have the advantages of high sensitivity and specificity, low requirement on hardware equipment, short reaction time, no complicated treatment on samples, suitability for on-site detection and the like, and are suitable for popularization and application.

Description

technical field [0001] The invention relates to a set of nucleic acid, a kit and a detection method for detecting Hantaan virus with RPA. The set of nucleic acid includes primers and probes, and belongs to the field of biotechnology. Background technique [0002] Hemorrhagic fever with renal syndrome (HFRS) is a natural source of disease caused by Hantavirus (HV), characterized by high fever, hemorrhage, hypotension, proteinuria and other renal dysfunction. Rats are the main source of infection. Inhalation of virus-contaminated aerosols, direct contact of skin or wounds with excrement and secretions of host animals, and eating food contaminated by host animal excreta can cause infection. There are about 60,000 to 100,000 cases of HFRS in the world every year, mainly in Eurasia, and the mortality rate is 3% to 10%. my country is the main epidemic area of ​​HFRS in the world, and the number of cases is about 50,000 to 80,000 per year, accounting for more than 80% of the world...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107C12Q2537/1376C12Q2521/507Y02A50/30
Inventor 韩一芳张锦海齐永汪春晖叶福强王太武张琪胡丹郑懿王长军
Owner 中国人民解放军东部战区疾病预防控制中心
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