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Triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus

A fluorescence quantification and reagent kit technology, applied in the biological field, can solve the problems of waste of reagent consumables, time-consuming and labor-intensive, etc.

Active Publication Date: 2014-01-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, single-tube real-time fluorescent quantitative RT-PCR detection of a pathogen is not only time-consuming and labor-intensive, but also wastes reagent consumables. Therefore, it is particularly important to establish multiple real-time fluorescent quantitative RT-PCR diagnostic methods and diagnostic reagents that can detect multiple pathogens at the same time.

Method used

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  • Triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus
  • Triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus
  • Triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus

Examples

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Effect test

Embodiment 1

[0047] see figure 1 , a triple fluorescent quantitative RT-PCR kit for detecting human calicivirus, characterized in that the kit consists of quantitative RT-PCR reaction solution tube 1, enzyme mixing solution tube 2, primer probe mixing solution tube 3, negative The control quality tube 4, three standard quality tubes 5, three positive control quality tubes 6 and the box body 7 are composed.

[0048] Wherein quantitative RT-PCR reaction solution tube 1 contains PCR reaction buffer, magnesium chloride and deoxyribonucleotide triphosphate mixture; enzyme mixture tube 2 contains heat-resistant Taq DNA polymerase, RNase inhibitor and MMLV reverse transcriptase; primer The probe mixture tube 3 contains specific primers for three types of Norovirus GI, Norovirus GII, and Zarovirus and corresponding three fluorescently labeled probes; three standard tubes 5 are respectively placed in Type GI Norovirus standard substance, GII type norovirus standard substance, and Zarovirus standa...

Embodiment 2

[0069] 1 Materials and methods

[0070] 1.1 Clinical specimens and viral nucleic acids:

[0071] The clinical samples of Norovirus GI, Norovirus GII, and Zarovirus were obtained from the stool or rectal samples of patients with diarrhea in the First Affiliated Hospital of Zhejiang University School of Medicine, the Children’s Hospital Affiliated to Zhejiang University School of Medicine, and several other hospitals in Zhejiang Province. Swab specimen, which is shipped to the laboratory after the sample is collected. All positive nucleic acids were confirmed by gene sequencing.

[0072] 1.2 Primers and probes

[0073]Multiple gene sequences covering Norovirus GI, Norovirus GII, and Zarovirus at home and abroad were downloaded from the NCBI gene bank in the United States. The homology comparison was carried out using DNAman software to determine the conserved regions of the above viral genomes. Primer Express 3.0 software was used to design highly specific primers and Taqman...

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Abstract

The invention discloses a triple fluorescent quantitative RT (Reverse Transcription)-PCR (Polymerase Chain Reaction) kit for detecting human calicivirus, which consists of a quantitative RT-PCR reaction liquid tube, an enzyme mixed liquid tube, a primer probe mixed liquid tube, a negative control sample tube, three standard sample tubes, three positive control sample tubes and a kit body. The standard sample is GI type norovirus, GII type norovirus and sapovirus standard samples. The control samples are positive samples and negative samples. According to the invention, specific primers and probes are designed on the basis of the sequence of a conserved region of the GI type norovirus, GII type norovirus and sapovirus and a one-step triple real-time fluorescent quantitative RT-PCR technique is adopted to accurately detect the GI type norovirus, GII type norovirus and sapovirus in a specimen. The kit is reasonably designed, simple and convenient to operate, quick and accurate.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a fluorescent quantitative RT-PCR kit for detection, in particular to a triple real-time fluorescent quantitative RT-PCR kit for one-step detection of human calicivirus. Background technique [0002] Human calicivirus (HuCVs) is one of the main pathogens causing non-bacterial acute diarrhea in children and adults, second only to rotavirus. It is highly contagious and spreads widely around the world, especially affecting the health of infants and young children. The earliest discovered human calicivirus was detected by the American scholar Kapikin in 1972 from the stool samples of gastroenteritis patients who had an outbreak in a school in Norwalk, Ohio, by immunoelectron microscopy, and named it Norwalk after the place of discovery. Wacker virus. Since then, human caliciviruses have been continuously discovered around the world. Viruses are classified according to the virus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 陈瑜李兰娟谢国良崔大伟
Owner ZHEJIANG UNIV
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