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Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting MERS (Middle East Respiratory Syndrome), and detection method thereof

A RT-PCR and fluorescent probe technology, applied in the field of early clinical diagnosis of Middle East Respiratory Syndrome Coronavirus, can solve the problems of increased epidemic risk and limited infectivity of MERS, so as to reduce the fatality rate, shorten the detection time, ensure The effect of reliability

Active Publication Date: 2015-08-19
深圳秀朴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On May 28, 2015, my country discovered and confirmed the first case of imported Middle East Respiratory Syndrome infection. Previous studies have shown that the infectivity of MERS is very limited. The virus may have been capable of continuous human-to-human transmission, and the risk of the epidemic has further increased. At the same time, due to the large-scale personnel exchanges between my country and the Middle East, such as business, religious exchanges, and tourism, there is a risk of further importation of the epidemic.
[0004] At present, there is no clear and effective anti-MERS-CoV drug, so controlling the further development of the epidemic mainly depends on timely detection and isolation

Method used

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  • Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting MERS (Middle East Respiratory Syndrome), and detection method thereof
  • Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting MERS (Middle East Respiratory Syndrome), and detection method thereof
  • Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting MERS (Middle East Respiratory Syndrome), and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The linear experiment of the first pair of probes of embodiment 1 kit

[0064] The first pair of target probes is labeled with FAM fluorescent groups. The linear reference product of the FAM channel of the kit consists of plasmids containing the first pair of target fragments. The positive plasmids are serially diluted 10 times with TE buffer. The following gradient solution was obtained (S1: 1.0 x 10 10 copies / ml, S2: 1.0×10 9 copies / ml, S3: 1.0×10 8 copies / ml, S4: 1.0×10 7 copies / ml, S5: 1.0×10 6 copies / ml, S6: 1.0×10 5 copies / ml, S7: 1.0×10 4 copies / ml, S8: 1.0×10 3 copies / ml, S9: 1.0×10 2 copies / ml,) as a linear reference for the FAM channel, the experiment shows that the kit is at 1.0×10 10 copies / ml to 1.0×10 3 There is a good linear relationship between copies / ml, the experimental results refer to figure 1 .

Embodiment 2

[0065] The minimum detection limit experiment of embodiment 2 kit

[0066] This experiment validates the lowest detection limit of the kit, using S6 in the linear reference product of the FAM channel: 1.0×10 5 copies / ml, S7: 1.0×10 4 copies / ml, S8: 1.0×10 3 Positive plasmids with equal concentrations of copies / ml are used as the detection limit reference product of the first pair of probes. This experiment shows that the detection limit of the kit reaches 5 copies / reaction. The experimental results refer to figure 2 .

Embodiment 3

[0067] Embodiment 3 The specificity experiment of kit

[0068] In this experiment, common pathogens with the same infection site as MERS-CoV were selected as specific reference substances, and inactivated clinical positive samples were used. Specific reference products are influenza A H1N1 virus, H7N9 avian influenza virus, influenza A H3N1 virus, influenza A H3N2 virus, norovirus type GI, norovirus type GII, parainfluenza virus type 1, parainfluenza virus Type 2, coronavirus 229E, enteric adenovirus, influenza virus type A. Use the universal virus RNA extraction kit developed by Shenzhen Yirui Biotechnology Co., Ltd. to extract the RNA in the above inactivated samples as a template for experiments. The experimental data shows that these 8 specific reference products do not have the typical "S" type The amplification curve shows that the specificity of the kit is good, and the experimental results refer to image 3 .

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Abstract

The invention discloses fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers and a fluorescent probe for detecting MERS (Middle East Respiratory Syndrome) coronavirus, which comprise a first primer pair, a second primer pair and a probe, wherein the first primer pair, second primer pair and fluorescent probe are a first specific forward / reverse primer pair, a second specific forward / reverse primer pair and a fluorescent probe which are respectively designed for conserved sequences of two different genes of the MERS coronavirus. The invention also discloses a kit and method for detecting the MERS coronavirus. By using the two specific probe pairs and primer which are designed for the conserved sequences of the two different genes of the MERS coronavirus, when the double-target PCR (polymerase chain reaction) detection result is positive, the infection of the MERS coronavirus in the clinical sample can be diagnosed. The method is simple, has the characteristics of high speed, high specificity and high sensitivity, and can be used for auxiliary diagnosis and epidemic situation monitoring of the MERS coronavirus.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and the invention provides a fluorescent RT-PCR RT-PCR primer, probe, kit and detection for detecting Middle East Respiratory Syndrome coronavirus (Middle East Respiratory Syndrome, MERS) in clinical samples The method can be used for clinical early diagnosis of MERS-CoV. technical background [0002] In September 2012, two cases of novel coronavirus infection with clinical manifestations similar to SARS were reported for the first time. On May 23, 2013, the World Health Organization (WHO) named the novel coronavirus infection as "Middle East Respiratory Syndrome (MERS)". From September 2012 to May 25, 2015, a total of 1139 confirmed cases of MERS were reported in 24 countries around the world, with 431 deaths and a case fatality rate of 37.8%. Among them, in the areas where the cases are concentrated in Saudi Arabia, as of the same day, Saudi Arabia has reported a total of 1,005 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6846C12Q1/701C12Q2521/107C12Q2563/107Y02A50/30
Inventor 朱海黄超杰游腾飞卢和华
Owner 深圳秀朴生物科技有限公司
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