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Annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method and application thereof

A technology for isothermal nucleic acid amplification and fluorescent probes, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. limited, broad application prospects, and the effect of a wide range of applications

Inactive Publication Date: 2019-08-06
SHANGHAI IGENETEC DIAGNOSTICS CO LTD +1
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  • Application Information

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Problems solved by technology

[0010] The TaqMan probe method commonly used in PCR has high specificity, but the BstDNA polymerase commonly used in the isothermal amplification reaction lacks the ability to cut the probe, so the high specificity detection method similar to the TaqMan probe is in the isothermal amplification reaction. Difficult to implement

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  • Annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method and application thereof
  • Annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method and application thereof
  • Annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method and application thereof

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Embodiment 1

[0037] The basic principle of embodiment 1 CFPA method

[0038] The present invention combines structure-specific endonuclease FEN1 with Bst DNA polymerase to develop a novel circular probe-mediated isothermal nucleic acid amplification method (CFPA). The basic principle of the method can be found in figure 1 .

[0039] Design a circular probe for the upstream and downstream of the target sequence, in which the 5' ends of the forward and reverse circular probes are labeled with a fluorescent group (F), and the nearby bases are labeled with a quencher group (Q) . In step (1), the second half of the forward fluorescent probe is first combined with the template and extended, and under the strand displacement action of Bst DNA polymerase, the first half of the probe is complementary to the sequence in the extension product, forming a circular structure at one end As shown in step (2). Step (3) When the reverse circular probe is paired with the template and extends to the circu...

Embodiment 2

[0040] Example 2 Verification of CFPA method reliability

[0041] 2.1 In order to verify the reliability of the CFPA method, we conducted the following experiments. Firstly, circular probes for rotavirus and astrovirus were designed, and plasmid molecules carrying conserved sequences of rotavirus and astrovirus were synthesized as positive controls, and ultrapure water was used as negative control. It was found that both virus plasmids could be successfully Amplified with good specificity (see respectively figure 2 A in and figure 2 in B).

[0042] Wherein, the nucleotide sequences of the forward circular fluorescent probe and the reverse circular fluorescent probe of rotavirus are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2; The 5' ends of the reverse circular fluorescent probes are all labeled with a FAM fluorescent group, and the 11th base of SEQ ID NO: 1 and the 12th base of SEQ ID NO: 2 are respectively labeled with a TAMRA quencher group.

[0043] The nucle...

Embodiment 3

[0062] Embodiment 3 utilizes CFPA method to detect rotavirus cDNA and RNA

[0063] 3.1 Assess the detection performance of the CFPA method for detecting rotavirus cDNA in actual samples. In 8 rotavirus positive and 24 rotavirus negative samples, CFPA maintained good specificity and sensitivity (respectively see image 3 A in and image 3 in B). The minimum detection limit of the CFPA method for detecting rotavirus cDNA was evaluated with serially diluted positive plasmid samples to 10copies / reaction (see image 3 C), and there is a good linear relationship between the Tt value and the logarithmic value (LogC) of the sample concentration (copies / μl) (see image 3 in D).

[0064] 3.2 The Bst 2.0 hot-start DNA polymerase used in the CFPA method also has natural reverse transcription activity, so this method has the ability to directly detect RNA samples.

[0065] Rotavirus is an RNA virus, and our direct CFPA assay using RNA extracted from clinical samples remained at 100% sp...

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Abstract

The invention relates to an annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method and an application thereof. The isothermal nucleic acid amplification method comprises the step of carrying out nucleic acid amplification of a to-be-detected nucleic acid sample under the action of structural-specificity endonuclease, DNA polymerase andan annular fluorescent probe. The annular fluorescent probe-mediated isothermal nucleic acid amplification method uses an FEN1 enzyme, realizes a signal monitoring mode of a probe method in the fieldof isothermal amplification, has high specificity and sensitivity, and has a good detection limit with the minimum detection limit of 10 copies / reaction. The method can detect DNA and RNA samples simultaneously, and has wide application scope; the diagnostic efficiency of the method is not significantly different from that of a traditional gold standard method, and the result shows that the annular fluorescent probe-mediated high-sensitivity and high-specificity isothermal nucleic acid amplification method provided by the invention has broad application prospects in nucleic acid analysis, clinical diagnosis and other fields.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and in particular relates to a high-sensitivity and high-specificity isothermal nucleic acid amplification method mediated by a circular fluorescent probe and its application. Background technique [0002] Nucleic acid detection is an important method widely used in biomedical research, and it has important clinical value in the application of infectious pathogen diagnosis. As the gold standard method in nucleic acid detection, PCR has the advantages of high specificity and sensitivity, but its operation is complicated and requires high requirements for instruments and personnel, which limits its application in bedside detection and on-site rapid detection . [0003] In order to overcome the shortcomings of the PCR method, a large class of isothermal nucleic acid amplification methods have emerged, such as recombinase polymerase amplification (RPA), loop-mediated isothermal am...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2521/301C12Q2521/319C12Q2521/101C12Q2563/107
Inventor 方雪恩叶辛孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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