Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses

An RT-PCR, hemorrhagic fever virus technology, applied in the field of fluorescence quantitative RT-PCR kits, can solve the problems of non-application, long identification time, delayed positive reaction, etc., to ensure accuracy and specificity, and the detection process is convenient. Fast, accurate results

Inactive Publication Date: 2012-04-25
中华人民共和国江苏出入境检验检疫局
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AI Technical Summary

Problems solved by technology

The cultivation of Xinjiang hemorrhagic fever virus requires a high level of biosafety in the laboratory, and it needs to be carried out in a BSL-3 laboratory. It takes a long time to isolate and identify the virus, and the detection sensitivity is not high; serological methods mainly include (1) complement fixation test : It mainly detects complement fixation antibody, the positive reaction appears later, and it is rarely used in clinical laboratories; (2) Neutralization test: The method is very complicated and generally not used in clinical practice; (3) Hemagglutination inhibition test, double serum needs to be collected Specimen, if the antibody titer of the second serum increases by more than 4 times, it is an indicator of recent infection
In addition, there are still reports using methods such as capture ELISA and indirect immunofluorescence to detect specific antibodies.
[0005] Compared with tissue culture and serological diagnosis, molecular biology methods have the advantages of high detection sensitivity, strong specificity and short detection time, but currently there is no fluorescent quantitative RT-PCR kit suitable for rapid detection of Xinjiang hemorrhagic fever virus at ports

Method used

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  • Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses
  • Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses
  • Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of positive control RNA template

[0040] According to the sequence of Xinjiang hemorrhagic fever virus (GenBank sequence number: HQ833035.1), a DNA fragment with a size of 606 bp containing the fluorescent RT-PCR detection fragment sequence was amplified, cloned into the PMD-18T vector, and purified with a plasmid DNA extraction kit Plasmid DNA, sequenced to confirm the orientation of the insert. Use universal primers paired with the vector to amplify the T7 promoter and the inserted Xinjiang hemorrhagic fever virus sequence, then use MAXIscript T7 Kit for in vitro transcription to generate target RNA, and then use TURBO DNase for DNase treatment to remove untranscribed PCR amplification. Increase the template, and finally use ethanol precipitation to purify the RNA product, which is the positive control RNA template, and is stored in a -80 °C ultra-low temperature refrigerator for later use.

[0041] Take 1 μL of in vitro transcribed template R...

Embodiment 2

[0043] Example 2: Determination of Amplification Procedures

[0044] The RT-PCR reaction kit was AgPath-ID? One-Step RT-PCR Kit (product of Ambion Company, USA), and the amplification and detection were carried out on ABI7500 real-time fluorescence quantitative PCR instrument.

[0045]According to the specific characteristics of primers and probes, the reaction program on the instrument tested 3 temperatures and 2 times in the selection of annealing extension conditions: 10 minutes at 45°C, 15 minutes at 95°C, and 40 PCR cycles: 15 minutes at 95°C seconds → (58 / 60 / 62) °C (30 / 45) seconds; the sample volume of each reagent is 1 μl of 25×RT-PCR enzyme mixture in each reaction tube, 2×RT-PCR reaction 12.5 μl buffer, 1 μl forward primer, 1 μl reverse primer, 0.5 μl fluorescent probe, 100 pg to 1 μg (≤ 10 μl) of template RNA, made up to a total volume of 25 μl with DEPC-treated water, and the fluorescence signal acquisition was set in the annealing extension stage . Positive ampli...

Embodiment 3

[0046] Embodiment 3: Determination of optimal primer concentration

[0047] When the final concentration of the probe was 250nM, the amplification test was carried out respectively by eight groups of forward and reverse primer concentrations listed in Table 2, and the amplification curve was obtained as a result of the test. figure 1 . from figure 1 Select the curve with the smallest Ct value and a more obvious amplification curve ( figure 1 The primer concentration corresponding to the third curve in ) is the optimal concentration, that is, the final concentration of the forward primer is 500nM, and the final concentration of the reverse primer is 750nM.

[0048] Table 2

[0049] group XHFV-FP / RP (final concentration nM) Ct value 1 750 / 500 13.25 2 750 / 250 13.57 3 500 / 750 13.18 4 500 / 500 13.34 5 500 / 250 13.71 6 250 / 750 13.66 7 250 / 500 13.27 8 250 / 250 13.65

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Abstract

The invention relates to a fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for rapidly detecting XHF (Xinjiang hemorrhagic fever) viruses. In the invention, a simple, rapid and sensitive RT-PCR detection kit is developed on the basis of designing a specific primer according to a specific conserved sequence of an XHF virus and successfully establishing a PCR detection method for XHF viruses. When XHF viruses are detected by using the kit and detection method provided by the invention, the detection process is convenient and rapid, a detection result can be obtained in 2-3 hours, and the detection sensitivity and the detection accuracy are high, therefore, the kit and detection method provided by the invention are especially suitable for the needs of entry-exit inspection and quarantine.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, in particular to a fluorescence quantitative RT-PCR kit for rapid detection of Xinjiang hemorrhagic fever virus, a detection method of the kit and the application of the kit in clinical and port detection of Xinjiang hemorrhagic fever virus. Background technique [0002] Xinjiang hemorrhagic fever (XHF) is an acute infection caused by Xinjiang hemorrhagic fever virus (XHFV, also known as Crimea-Congo hemorrhagic fever virus) belonging to the genus Neirovirus in the family Buniaviridae. The disease, which is transmitted by tick vectors, is mainly prevalent in Europe, Asia and Africa. The occurrence of hemorrhagic fever in Xinjiang has obvious seasonality. The peak of the epidemic is from April to May every year, which is related to the rise and fall of ticks in nature and the busy pastoral activities. match the season. Clinical features include fever, headache, drowsiness and fatigue, v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 孙立新丁永健胡红霞朱临朱光耀朱国强杨庆贵
Owner 中华人民共和国江苏出入境检验检疫局
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