Real-time PCR detection method for human group H rotavirus

A quantitative detection method and the technology of grouping rounds, applied in the field of microorganisms, can solve the problems that hinder the development of vaccine technology and the lack of detection methods in the epidemic situation of group H rotaviruses, and achieve convenient application, high detection sensitivity and specificity, and good specificity Effect

Pending Publication Date: 2021-06-01
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no method to detect group H rotavirus, the lack of detection methods, and few reports on grou

Method used

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  • Real-time PCR detection method for human group H rotavirus
  • Real-time PCR detection method for human group H rotavirus
  • Real-time PCR detection method for human group H rotavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] In order to realize the human H group rotavirus infection and culture of the passaged cells, firstly, the passaged cells are cultured. Passage cells include but are not limited to MA104 cells. The following takes human group H rotavirus J19 virus infection and culture of MA104 cells as an example to illustrate.

[0061] 1.1 Culture MA104 cells

[0062] 1.1.1 Recovery of cells

[0063] (1) Take out the required cell cryopreservation tube from the liquid nitrogen tank, put it into warm water at 37°C and shake it back and forth to melt it quickly, and centrifuge at 800rpm / min for 3min.

[0064] (2) Pour off the cell freezing solution.

[0065] (3) Prepare cell culture medium containing 10% FBS, that is, 1mL FBS+9mL1640, and the actual dosage is calculated according to the specific amount required for each experiment.

[0066] (4) Draw 6mL of cell culture solution into a 25cm 2 of cell flasks.

[0067] (5) at 25cm 2 Pipette 1mL of cell culture medium into the cell fl...

Embodiment 2

[0090] Example 2 Real-time PCR Quantitative Detection of Human Group H Rotavirus

[0091] 1. Preparation of RNA Standards

[0092] (1) Extraction of J19 virus RNA

[0093] Take 200 μl rotavirus J19 strain virus liquid, according to the QIAGEN company The viral RNA mini kit was operated according to the instructions, and the viral RNA genome was extracted and stored at -80°C.

[0094] (2) RT-PCR for one-step amplification of the target gene of the standard item

[0095] With primers SEQ ID NO:13 and SEQ ID NO:14, J19 viral RNA was subjected to one-step RT-PCR amplification, and the primer sequences are shown in Table 1. Amplify the VP6 gene fragment of Group H rotavirus J19 strain, the size is 657bp, and the primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0096] Table 1

[0097] Primer serial number Nucleotide sequence upstream primer SEQ ID NO: 13 ATGGAGCAGCAACTAGAACC downstream primer SEQ ID NO: 14 TACAACATTAGGTGG...

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Abstract

The invention belongs to the technical field of microorganisms, and discloses a Real-time PCR quantitative detection method for human group H rotaviruses. An upstream primer F for Real-time PCR quantitative detection comprises SEQ ID No.1 and a sequence which at least has 70% of homology with the SEQ ID No.1; a downstream primer R comprises SEQ ID No.2 and a sequence which has at least 70% of homology with the SEQ ID No.2; and the probe comprises SEQ ID No.3 and a sequence which has at least 70% of homology with the SEQ ID No.3. The Real-time PCR circulation conditions are as follows: reverse transcription is performed for 5-8 minutes at 48-55 DEG C, PCR initial activation is performed for 20-50 seconds at 95 DEG C, denaturation is performed for 10-20 seconds at 95 DEG C, annealing and extension are performed for 50-70 seconds at 55-65 DEG C, 35-45 cycles are performed, and a fluorescence signal is detected after each cycle. The method can be used for rapidly and effectively detecting the human group H rotavirus infection. The detection method provided by the invention has relatively high detection sensitivity and specificity, is simple to operate and convenient to apply, and provides feasible technical support for group H rotavirus monitoring and group H rotavirus infection disease prevention and control in the fields of clinical diagnosis, inspection and quarantine and the like.

Description

technical field [0001] The invention belongs to the technical field of microbes, and relates to a Real-time PCR detection method for human group H rotavirus. Background technique [0002] Rotaviruses (RVs) belong to the genus Rotavirus in the Reoviridae family and are mainly transmitted through the fecal-oral route. They are the main pathogens that cause severe diarrhea in infants and young children worldwide. According to statistics, more than 2 million children with diarrhea are hospitalized due to rotavirus infection every year, and about 500,000 infants and young children die due to rotavirus infection. Currently, there is no specific drug for the treatment of rotavirus diarrhea. Generally, symptomatic treatment is used. Vaccination can play a protective role and is an effective means to prevent rotavirus infection. The continuous emergence of new circulating strains of rotavirus has brought great challenges to the development and use of rotavirus vaccines. [0003] Ni...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2545/114C12Q2561/113C12Q2563/107
Inventor 段招军庞立丽李丹地
Owner 中国疾病预防控制中心病毒病预防控制所
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