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Method for simultaneously detecting three groups of rotaviruses A, B and C

A group A rotavirus, virus technology, applied in the simultaneous detection of A, C three groups of rotavirus, B field, can solve the problems of false negative, low detection sensitivity, different amplification efficiency, etc., to achieve strong specificity and sensitivity High, short detection time effect

Inactive Publication Date: 2020-01-21
山东凯景生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult for the traditional multiplex PCR technique to simultaneously amplify multiple target sequences with multiple pairs of primers, because each different target sequence has its own optimal amplification conditions, and it is difficult to unify them. Even if unified, each target sequence There are also different amplification efficiencies, resulting in low concentration / titer pathogens not being amplified and not detected, resulting in low detection sensitivity and false negative results

Method used

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  • Method for simultaneously detecting three groups of rotaviruses A, B and C
  • Method for simultaneously detecting three groups of rotaviruses A, B and C
  • Method for simultaneously detecting three groups of rotaviruses A, B and C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 Primer probe design and synthesis

[0059] A pair of specific primers, a specific probe with different fluorescent labels and a pair of universal primers were designed respectively for group A rotavirus, B rotavirus and group C rotavirus.

[0060] Table 1 List of detected pathogens

[0061] serial number virus name English abbreviations target gene 1 group A rotavirus RV-A VP6 2 group B rotavirus RV-B VP6 3 Group C rotavirus RV-C VP6

[0062] Multiple gene sequences covering domestic and foreign RV-A, RV-B, and RV-C viruses were downloaded from the NCBI gene bank. The homology comparison was carried out using BioEditor software to determine the conserved regions of the above viral genomes. Primer Premier 5.0 software was used to design highly specific primers and probes in its conserved regions. The sequences of primers and probes were verified by Blast and had good specificity. Label the VIC fluorophore ...

Embodiment 2

[0065] Example 2 Sensitivity Evaluation of Rotavirus Multiplex PCR Detection Method

[0066] 1. Preparation of samples to be tested:

[0067] The construction method of RV-A plasmid: the nucleic acid of group A rotavirus samples is amplified by PCR method using specific primers (i.e. RV-A specific primers in the present invention) to obtain group A rotavirus target gene fragments After purification, it was cloned into the pMD18-T vector by TA, identified by sequencing, and the recombinant vector with the correct sequencing result was transformed into DH5α competent cells, amplified, and RV-A plasmid was obtained.

[0068] The construction method of RV-B plasmid: apply specific primers (i.e. RV-B specific primers in the present invention) and use PCR method to amplify the nucleic acid of group B rotavirus samples to obtain group B rotavirus target gene fragments After purification, it was cloned into the pMD18-T vector by TA, identified by sequencing, and the recombinant vecto...

Embodiment 3

[0089] Example 3 Rotavirus multiplex PCR detection method detection effect evaluation

[0090] 1. Sample processing

[0091] Two cases of stool samples from patients confirmed positive for RV-A, RV-B, and RV-C in clinical testing, and two cases of serum samples from healthy people were selected, and a total of 8 samples were extracted for RNA extraction. Commercial kits can be used to extract RNA, such as using QIAGEN ViralRNA Mini Extraction Kit (CAT: 52904) to extract viral RNA. The extraction steps are well known to those skilled in the art and will not be repeated here.

[0092] 2. PCR amplification reaction

[0093] A total of 8 viral RNA samples extracted above were selected, plus a negative control / positive control reaction, and a total of 10 tubes of PCR reactions were required. Add the viral RNA samples to be tested to reaction tubes 1 to 8, and add 1×10 to tube 9. 6 Copies / ml plasmid positive control substance, add DEPC-ddH2O to No. 10 reaction tube as negative co...

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Abstract

The invention discloses a method for simultaneously detecting three groups of rotaviruses A, B and C, and belongs to the field of biological detection. A pair of specific primers and a specific probewith different fluorescent labels are designed for target genes of group A rotavirus, group B rotavirus and group C rotavirus, a pair of universal primers is further designed, and during PCR amplification, the specific primers with universal primer tags are used for enrichment and amplification; and then universal primer tags are used for exponential amplification, and fluorescence signals of exponential amplification products are collected for detection. The method can simultaneously detect the group A rotavirus, the group B rotavirus, and the group C rotavirus. The method is high in specificity, high in sensitivity and short in detection time, and can be used for early rapid diagnosis of rotavirus infection and epidemiological study of the rotavirus.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for simultaneously detecting three groups of A, B and C rotaviruses. Background technique [0002] Rotavirus (rotavirus, RV) is the main pathogen that causes infantile diarrhea and animal viral diarrhea in autumn and winter. It belongs to the genus Rotavirus in the Reoviridae family and is a double-stranded RNA virus. Rotavirus is not only an important cause of severe diarrheal diseases in infants, children and adults, but also an important cause of diarrheal diseases in young and adult mammals and poultry. It is widely prevalent in the world and causes huge losses every year. At present, it is divided into 8 groups, numbered in English letters as A, B, C, D, E, F, G and H. Among them, three groups A, B, and C can be pathogenic to humans. Group A rotavirus is the most common and most harmful, and is one of the main pathogens causing diarrhea in infants and young chil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16
Inventor 靖相密张通刘奕邓雪萍
Owner 山东凯景生物技术有限公司
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