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Detecting method for RNA isothermal transcription amplification and its reagent kit

A detection method and RNA polymerase technology are applied in the improvement field of RNA amplification detection technology, and can solve the problems of complicated operation, inability to achieve quantitative detection level, expensive detection instruments and the like

Inactive Publication Date: 2007-04-11
SHANGHAI FOSUN PHARMA (GROUP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to provide a kind of RNA isothermal transcription amplification and nano-gold probe detection method (Nucleic Acid Isothermal and Transcriptional Amplification with GoldNano-particle Probes, be called for short NITAG) and kit thereof, to overcome NASBA, TMA and Methods such as LAMP are cumbersome to operate or require expensive detection instruments, and at the same time cannot achieve quantitative detection, and the detection is convenient

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  • Detecting method for RNA isothermal transcription amplification and its reagent kit
  • Detecting method for RNA isothermal transcription amplification and its reagent kit
  • Detecting method for RNA isothermal transcription amplification and its reagent kit

Examples

Experimental program
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Effect test

Embodiment 1

[0073] NITAG detection of RNA samples

[0074] The implementation of the present invention is illustrated below by introducing the detection of hepatitis C virus (HCV) RNA samples.

[0075] Take 100 μL of HCV clinical serum-positive samples, and extract HCV RNA samples according to the instructions of Trizol (Invitrogen, USA) reagent.

[0076] Design of primers and probes: According to literature reports and HCV nucleotide sequence alignment, design isothermal amplification primers and alkylthiolated oligonucleotide probes in the relatively highly conserved 5′-UTR region, upstream primer: 5′ -GAG TGT CGT GCA GCC TCC A-3', downstream primer: 5'- AAT TCT AAT ACG ACT CAC TAT AGG G CAC TCG CAA GCA CCC TAT CA-3', where the underline is the promoter sequence that can be recognized by T7 RNA polymerase; 5'-end thiolated oligonucleotide probe: 5'-HS-(CH 2 ) 6 -[TAC ACC GGA ATT GCC AGG ACG AC]-3’; 3’-Hexylthiolated oligonucleotide probe: 5’-[TGG GTC GCG AAA GGC CTT GTG G]-(CH 2 ...

Embodiment 2

[0081] Adopt the HCV detection kit (A) that assembles according to the present invention, detect 20 HCV positive serum samples and 20 negative serum samples in clinical, simultaneously with the HCV detection kit (B) of Gen-Probe company based on TMA technology For detection comparison, the operation method of the kit of the present invention is according to Example 1, the kit of Gen-Probe Company is stored at -70°C, the operation is carried out according to the instructions and detected by a chemiluminescent detection instrument, and the results are shown in Table 1.

[0082] The overall coincidence rate of the results of the method of the present invention and the TMA technique reached 97.5% in 40 samples, and the results were basically consistent. Wherein 20 routine HCV negative serum samples both detection results are identical; For 20 routine HCV positive serum samples, 19 routine both results meet, 1 routine is positive reagent of the present invention, TMA technology reag...

Embodiment 3

[0090] Adopt the HCV detection kit (A) that assembles according to the present invention, detect 20 parts of HCV positive serum samples and 20 parts of negative serum samples in the clinic, obtain the fluorescent RT-PCR HCV detection kit (C) of domestic drug certificate simultaneously ) for comparison, the operating method of the kit of the present invention is according to Example 1, the control kit (C) is operated according to the instructions, and finally detected on a real-time fluorescent PCR instrument, the results are shown in Table 2, based on the principle of amplification and detection are different According to the technology of the present invention and real-time fluorescent RT-PCR technology, the overall coincidence rate of detecting 40 cases of HCV serum samples is 92.5%, and the results are basically consistent.

[0091] The HCV detection comparative result of table 2 kit of the present invention and fluorescence RT-PCR control technology

[0092]

[0093] Po...

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Abstract

The present invention relates to one kind of RNA isothermal transcription amplification detection process and its reagent kit. The detection process includes the following successive steps: delinking RNA template at 60-70 deg.c and cooling; adding reverse transcriptase, ribonuclease H and RNA polymerase, and isothermal amplification at 37-42 deg.c under the guide of primer to obtain RNA amplicon; detecting RNA amplicon with the oligonucleotide marked nanometer metal probe and color reaction to obtain detection result. The hybrid solution, microporous board or test paper is blue for positive detection result or red for negative. The present invention has the advantages of being simple, fast, sensitive, etc and may be used in RNA detection widely in experimental research, clinical detection, food safety detection, medicine screening, etc.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to the improvement of RNA amplification detection technology. Background technique [0002] In the process of nucleic acid detection, in many cases, the content of the nucleic acid to be tested in the sample is relatively low. In order to achieve a certain sensitivity and accuracy in the detection, it is necessary to effectively amplify the nucleic acid fragments to be tested in the sample. Since the United States Cetus and the University of California jointly created the Polymerase Chain Reaction (PCR) in 1985, it has greatly promoted the development of nucleic acid detection technology and even the entire molecular biology technology, and has gradually become a nucleic acid detection technology. It is an important means in the process and also becomes the basis of modern molecular biology technology. Subsequently, a series of nucleic acid amplification detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 吴大治夏懿沈维祥
Owner SHANGHAI FOSUN PHARMA (GROUP) CO LTD
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