Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit

A technology of fluorescence quantification and standard products, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome operation and low detection sensitivity, and achieve the effect of improving efficiency

Inactive Publication Date: 2013-06-12
GUANGZHOU VIPOTION BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It uses RNA as a template to reverse transcribe the first strand of complementary DNA (cDNA), and then uses this strand as a template to carry out PCR reaction, so that it can detect the specific detection of viral nucleic acid with a very low copy number in the sample, overcoming the traditional The method has the disadvantages of low detection sensitivity and cumbersome operation

Method used

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  • Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit
  • Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit
  • Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1: Collection and delivery of specimens

[0119] The types of specimens suitable for testing include stool specimens from suspected patients, as well as specimens such as throat swab specimens, herpes fluid, cerebrospinal fluid, or virus isolation and culture.

[0120] Stool specimen collection and transportation methods: Swab feces or diarrhea with a sterile swab, put them in a sterile container, seal it and send it to the laboratory for testing immediately, and wrap the specimen in dry ice during transportation; or store it at -20°C for a short period of time. Long-term storage can be placed at -70°C, in principle, it should not exceed 6 months to prevent the degradation of viral RNA.

[0121] The method of collecting and transporting the throat swab specimens: swab the secretions from the posterior pharyngeal wall and tonsils on both sides of the throat swab under aseptic conditions, put them into the sampling tube, seal it and send it to the laboratory for t...

Embodiment 2

[0125] Embodiment 2: Extraction of viral RNA in the specimen

[0126] Take 100 μl of the sample solution collected above and add 300ul of the RNA extraction solution in the kit to fully shake, and place at room temperature for 5 minutes; add 100ul of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 5 minutes, and centrifuge at 13,000rpm at 4°C for 10 minutes; carefully transfer the upper aqueous phase to Add an equal volume of isopropanol to a clean centrifuge tube, mix thoroughly, and centrifuge at 13,000rpm for 10min; discard the supernatant, add 500μl 75% DEPC ethanol, mix well, centrifuge at 13,000rpm for 10min, and carefully absorb most of the ethanol; The extraction tube was left open and dried in air at room temperature for 5 min until the ethanol evaporated, and the precipitate was dissolved with 20 μl DEPC H2O. Take 50 μl negative quality control product and add 300 μl Trizol RNA extraction solution to shake fully, let it stand at room te...

Embodiment 3

[0127] Example 3: Preparation of HRVA-NVGⅠ-NVGⅡ-HAstV single standard

[0128] 3.1 RT-PCR experiment, cDNA synthesis by reverse transcription

[0129] Using the above extracted RNA as a template, prepare a reverse transcription reaction system according to PrimeScript® 1st Strand cDNA Synthesis Kit (TAKARA) instructions, the total system is 20ul, and synthesize the first strand of cDNA:

[0130] Random primer 1.0ul dNTPs (10mM) 1.0ul RNA 5.0ul RNase free dH 2 o 3.0ul Total 10.0ul

[0131] The above reaction solution was cooled at 65°C for 5 minutes, and then cooled on ice.

[0132] The above reaction solution 10.0ul 5×PrimerScript Buffer 4.0ul RNase inhibitor (40U / ul) 0.5ul PrimerScipt Rtase (200U / ul) 1.0ul RNase free dH 2 o 4.5ul Total 20.0ul

[0133] 30°C, 10min; 42°C, 60min; 70°C, 15min, synthesize the first strand of cDNA, obtain HRVA, NVGⅠ, NVGⅡ and HAstV cDNA templates, and collect th...

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Abstract

The invention discloses a single standard product-based quardruple four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) technology. The technology is capable of detecting group A rotavirus (HRVA), norovirus group I (NVGI), norovirus group II (NVG II) and human astrovirus (HAstV) RNA in various treated specimen through one-step quardruple PCR amplification and quantifying virus load. The technology is characterized in that single standard product is adopted as positive control and quantitative standard in the quardruple four-color fluorogenic quantitative PCR detection for four viruses, thereby breaking through the deficiency that the traditional multiple fluorogenic quantitative PCR needs a plurality of standard products, improving the experiment detection efficiency, reducing the pollution probability and improving the preparation and production efficiency of the kit. The method and kit can be used for fast high-throughput test and epidemic monitoring in laboratories of HRVA, NVG I, NVG II and HAstV which can cause diarrhea syndromes. The single standard product-based four-color florogenic quantitative PCR method and the kit belong to the biotechnology field.

Description

technical field [0001] The invention relates to virus nucleic acid detection and belongs to the field of biotechnology. The present invention adopts the four-fold four-color fluorescent quantitative PCR technology based on a single standard, and detects group A rotavirus (HRVA), norovirus group I (NVGI), and norovirus in various processed specimens through one-step PCR amplification. Such as virus group Ⅱ (NVGⅡ) and human astrovirus (HAstV) RNA, used for laboratory rapid detection and outbreak of rotavirus, norovirus group Ⅰ, norovirus group Ⅱ and human astrovirus that cause diarrhea syndrome monitor. The present invention also puts forward higher requirements on the design of primers and probes and the implementation of experiments. Background technique [0002] WHO estimates that there are 3 to 5 billion cases of diarrhea every year in the world, and 500,000 to 1,000,000 cases die from severe diarrhea, with an average of 25,000 deaths per day, especially in children. Cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 刘健翊周孟陈瑶王宝玉廖杰叶奕东黄佳柳莫海月徐德峰黄常艳徐贵云
Owner GUANGZHOU VIPOTION BIOTECH
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