High-sensitivity real-time fluorescence detection method

A technology for detecting samples and target nucleic acids, applied in the field of nucleic acid detection, can solve the problems of lower detection value, lower fluorescence ΔR value, lower detection sensitivity, etc.

Active Publication Date: 2012-07-04
苏州华益美生物科技有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] However, the inventors have found that in the practical application of real-time PCR, the Taqman probe will increase the fluorescence background value of the entire detection, resulting in a decrease in the fluorescence ΔR value, thereby reducing the sensitivity of detection; , although it can effectively reduce the fluorescence background value, but at the same time it will reduce the detection value, which will also reduce the ΔR value, and the reduction will even exceed the use of Taqman probes, which may further reduce the detection sensitivity

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Embodiment 1

[0039] The detection of embodiment 1 DNA sequence

[0040] We designed primers and probes for the HBV S gene sequence, and entrusted Beijing Liuhetong Economic and Trade Co., Ltd. to synthesize the following PCR amplification primers and probes (including control probes) to contain HBV (hepatitis B virus nucleic acid (HBV DNA) ) Qualitative and quantitative national reference material, No. 300009, can be purchased from China National Institutes for Food and Drug Control according to regulations, diluted with purified water) 1 × 10 2 IU / ml (positive), 20IU / ml (sensitivity 1) and 10IU / ml (sensitivity 2) aqueous solution or pure water (negative) are respectively used as templates, and are carried out on ABI 7500 fluorescent PCR instrument (available from Applied Biosystems company) PCR detection:

[0041] Forward primer: CGAGGCAGGTCCCCTAGAA

[0042] Reverse primer: CGGCGATTGAGACCTTCGT

[0043] The probe (control probe 1) used in the traditional Taqman-specific method: FAM-AGAA...

Embodiment 2

[0052] The detection of embodiment 2RNA sequence

[0053] We designed primers and probes for the HCV NS2 gene, and commissioned Beijing Liuhetong Economic and Trade Co., Ltd. to synthesize the following PCR amplification primers and probes (including control probes) to contain HCV (the second-generation HCV RNA national reference product, No. 300012, can be purchased from China National Institutes for Food and Drug Control according to regulations, diluted with purified water) 5 × 10 2 IU / ml (positive), 2.5×10 2 IU / ml (sensitivity 1) and 1×10 2 The aqueous solution (positive) or water (negative) of IU / ml (sensitivity 2) is respectively used as template, carries out RT-PCR detection on ABI 7500 fluorescent PCR instrument (available from Applied Biosystems company):

[0054] Forward primer: TCGCCATATTACAAGCGCTACA

[0055] Reverse primer: GCGCTTCTACTCTGGTCAGAAAA

[0056] The specific probe (control probe 1) used in the traditional Taqman method: FAM-CATGTGGTGGCTTCA-ECLIPSE, t...

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Abstract

The invention discloses a high-sensitivity real-time fluorescence detection method. The invention provides a method for detecting a target nucleic acid in a detected sample, which comprises: mixing the sample with a polymerase chain reaction(PCR) amplification agent and a fluorescence labeling probe, wherein the nucleic acid part of the fluorescence labeling probe comprises a first complementary sequence, a target nucleic acid specific sequence, a second complementary sequence, and an extension sequence, and the first complementary sequence and the second complementary sequence can complementand match with each other; and performing PCR and real-time fluorescence detection. The invention also provides a detection kit, use of the detection kit in preparation of a detection product, and primers, probes and other reagents used in the detection kit.

Description

Technical field: [0001] The invention belongs to the field of nucleic acid detection, in particular, the invention relates to a high-sensitivity real-time fluorescence detection method and detection kits and reagents used therein. Background technique: [0002] The Taqman probe (Taqman Probe) and molecular beacon technology (Molecular Beacon) invented in the 1990s greatly promoted the application of fluorescent real-time polymerase chain reaction (PCR) technology in nucleic acid detection And development. In real-time PCR detection, a specific nucleic acid labeled with a fluorescent group and a quencher group is usually used as a probe to implement real-time detection during the PCR amplification process. For example, Chinese patent document CN101131347A discloses a fluorescent quantitative PCR kit for rapid detection of Schistosoma japonicum, including forward primers, reverse primers and fluorescent probes, which are used for fluorescent quantitative PCR to detect the DNA...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 张文艳陈悦科刘明霞
Owner 苏州华益美生物科技有限公司
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