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Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof

A detection kit and real-time fluorescence quantitative technology, applied in the field of life, can solve the problems of wasting clinical specimens and reagents, time-consuming and labor-intensive expenses, and achieve the effect of reducing mortality and sequelae

Active Publication Date: 2011-10-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Given the increasing importance of human rotavirus, saruvirus, adenovirus, astrovirus and norovirus in public health, it is time-consuming, laborious and wasteful to simultaneously test for multiple diarrhea viruses in diarrhea surveillance work Clinical Specimens and Reagents
Compared with the PCR method, virus culture is time-consuming, laborious and costly, and there are no mature culture methods for Sars virus and Noro virus in the world.
At present, because the detection methods of rotavirus and norovirus are relatively more and more mature, while the research on the detection methods of rotavirus, adenovirus and astrovirus is relatively small, especially the method of multiplex fluorescent quantitative PCR.

Method used

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  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof
  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof
  • Triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit and use thereof

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Embodiment 1

[0050] see figure 1 , a triple real-time fluorescence quantitative RT-PCR detection kit, including: quantitative RT-PCR reaction solution, Zyzavirus standard, Astrovirus standard, Adenovirus standard, positive control, negative control, box body 7. There are container holes for placing the quantitative RT-PCR reaction solution tube 1, the standard sample tube 2 of Zaravirus, the standard tube 3 of astrovirus, the 4 standard sample of adenovirus, the positive control tube 5, and the negative control tube 6. , wherein the quantitative RT-PCR reaction solution is packed in a single tube, arranged in a matrix, and the standard products are gene standards of Sarsovirus, Astrovirus and adenovirus, wherein the fluorescent quantitative RT-PCR reaction solution contains RT-PCR reaction buffer (containing Magnesium chloride and deoxyribonucleotide triphosphate mixture, etc.) 3 tubes (labeled as ), three virus universal primers (upstream and downstream primers in the same tube) are 3 ...

Embodiment 2

[0052] 1 Materials and methods

[0053] Virus strains and clinical specimens:

[0054] The positive nucleic acids of enteric adenovirus 40 / 41, astrovirus and saruvirus (all identified by gene sequencing) were from the First Affiliated Hospital of Zhejiang University School of Medicine. Clinical samples were obtained from patients' stool samples, which were collected and transported to the laboratory on ice.

[0055] 1.2 Primers and probes

[0056] Downloaded the gene sequences of Sarsovirus, Astrovirus, and Adenovirus from all over the world from the NCBI Gene Bank in the United States. The homology comparison was carried out, and specific primers and Taqman probes were designed in the conserved gene region corresponding to the virus genome. The sequence is as follows:

[0057] Upstream primer binding such as virus-FP: 5'- CAACTATGACCAGGCTCTCGC-3'

[0058] Downstream primer binding such as virus-RP: 5'- GCCCTCCATYTCRAACACTA-3'

[0059] Specific probes such as virus-P: 5'-...

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Abstract

The invention provides a triple fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) detection kit, which comprises quantitative RT-PCR reaction liquid, standard substances and reference substances. A kit body is provided with container holes in which quantitative RT-PCR reaction liquid tubes, a Sapovirus standard substance tube, an astrovirus standard substance tube, an adenovirus standard substance tube, a positive reference substance tube and a negative reference substance tube are arranged respectively, wherein the quantitative RT-PCR reaction liquid tubes are separately arranged and arrayed in a matrix; the standard substances are standard substances for Sapovirus, astrovirus and adenovirus; and the reference substances are positive and negative reference substances. By a real-time fluorescence quantitative RT-PCR technology, three kinds of virus specific primers and specific fluorescent probes are adopted, the design is reasonable, the kit can detect the Sapovirus, astrovirus and adenovirus from a sample through PCR reaction, and the detection method is simpler, quicker and more accurate.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a fluorescent quantitative RT-PCR detection kit, in particular to a triple (three probes) real-time fluorescent quantitative RT-PCR in one reaction tube for simultaneous detection of sputum in feces samples of patients with gastroenteritis and diarrhea. For example, virus, astrovirus and adenovirus nucleic acid and detection methods can be applied to laboratory emergency detection of outbreaks caused by savirus, astrovirus and adenovirus. Background technique [0002] Diarrheal disease is a common and frequently-occurring disease affecting human health worldwide, especially one of the main causes of morbidity and mortality in infants and young children. Studies have shown that more than 2 million infants die from diarrhea-related diseases and their complications every year, and more than 80% of the patients come from underdeveloped countries. Although at least 25 different bac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 陈瑜李兰娟崔大伟
Owner ZHEJIANG UNIV
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