Isothermal nucleic acid amplification system with high specificity as well as application thereof

An isothermal nucleic acid amplification and high-specificity technology, applied in the field of warm nucleic acid amplification system and kits of warm nucleic acid amplification system, can solve the problems of limiting clinical practical application, lack of annealing temperature, false positives, etc., and achieve accurate expansion Rapid detection, broad clinical significance, and high specificity

Active Publication Date: 2018-11-06
SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these isothermal nucleic acid amplification methods often lead to serious false positives due to the complex composition of primers, high concentration and lack of uniform annealing temperature, which limits their clinical practical application.

Method used

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  • Isothermal nucleic acid amplification system with high specificity as well as application thereof
  • Isothermal nucleic acid amplification system with high specificity as well as application thereof
  • Isothermal nucleic acid amplification system with high specificity as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1—Verification of the basic principle of the Exo-NAT method

[0033] In order to verify the reliability of the mechanism of the Exo-NAT method in this example, the following experiment was adopted: first, the 5' end of the internal primer (FIP / RIP) was labeled with fluorescent groups FAM and HEX, and the 11th base T and The quencher group TAMRA is marked on the 12th base T of RIP to form FAM-FIP and HEX-RIP. When the exonuclease activity works in the reaction, the two fluorescent groups will be cleaved and combined with the quencher group separation, and thus fluorescence growth, as figure 2 The results of parts A and B in the middle test the above hypothesis and prove the reliability of the reaction mechanism; in addition, the method of agarose gel electrophoresis was used to analyze the distribution of the amplified product, and the results were as follows: figure 2 As shown in parts C and D of the three viruses, two bands appeared in the positive sample...

Embodiment 2

[0034] Example 2—Applying the Exo-NAT method to the detection of pathogens in children with diarrhea

[0035]In this example, the Exo-NAT method was used to detect rotavirus, astrovirus and adenovirus, and the embedded fluorescent dye (SYTO-9) combined with melting curve analysis was used to successfully realize the specific detection of the above three viruses.

[0036] In the embodiment, the reaction amplification system is 25 μL, and the concentration of each component is: 20mM Tris-HCl (pH8.8@25℃), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 8mM MgSO 4 , 0.1% Tween-20, 0.2μΜ FOP / BOP, 1.6μΜ FIP / RIP, 5U full-length Bst DNA polymerase, dNTPs (each 1.4mM), 1×SYTO-9, the reaction conditions are: 65°C, 90mins; where, The primers used to detect group A rotavirus are FOP, ROP, FIP, RIP primers shown in SEQ ID NO.1~SEQ ID NO.4; the primers used to detect astrovirus are as shown in SEQ ID NO.5 ~FOP, ROP, FIP, RIP primers shown in SEQ ID NO.8; primers for detecting adenoviruses are FOP, ROP,...

Embodiment 3

[0038] Example 3—Applying the Exo-NAT method to the multiple detection of pathogens in children with diarrhea

[0039] In this embodiment, the primer sets of the three viruses described in Example 2 are mixed in one system, which can realize multiple detection with high specificity for each virus, and the results are as follows Figure 5 shown, can pass T m The different values ​​can accurately determine the type of pathogens infected. In addition, there are two clinical specimens with double infection of rotavirus and adenovirus, and two characteristic T m The peak value shows that the Exo-NAT method described in this application has the ability of multiple detection. The minimum detection limit of the method was further evaluated for multiple detection, and the results showed that the minimum detection limit was still 100copies / reaction (such as Figure 6 ), which proved that the method has good multiple detection ability.

[0040] The amplification system and Exo-NAT met...

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Abstract

The invention relates to an isothermal nucleic acid amplification system with high specificity. The isothermal nucleic acid amplification system with high specificity comprises overall-length Bst DNApolymerase and also comprises embedded fluorescent dye. The invention also relates to a kit containing the isothermal nucleic acid amplification system as well as the application of the isothermal nucleic acid amplification system to childhood diarrhea pathogen detection. In the application, amplification primers comprise primer groups which are as shown in SEQ ID NO.1 to SEQ ID NO.4, SEQ ID NO.5to SEQ ID NO.8 and SEQ ID NO.9 to SEQ ID NO.12 and are correspondingly used for detecting Group A rotaviruses, astroviruses and adenoviruses. The overall-length Bst DNA polymerase has 5'-3'-duplex-specific excision enzyme activity and polymerization activity, the characteristic is applied to the Exo-NAT method of the invention, the embedded fluorescent dye is added into the reaction system and melting curve analysis is cooperated, so that high-specificity and high-sensitivity multiple nucleic acid detection can be realized; and the isothermal nucleic acid amplification system is successfully applied to accurate detection of childhood diarrhea pathogens, and has an important clinical significance and a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid amplification, and in particular relates to an isothermal nucleic acid amplification system with high specificity, a kit containing the isothermal nucleic acid amplification system and an application thereof. Background technique [0002] Infectious diarrhea in children is a clinically frequently-occurring and common disease that seriously threatens children's lives and health. It ranks among the top three causes of child mortality all year round, and the disease is more serious in developing countries. There are many types of pathogens that cause infectious diarrhea in children. Among them, group A rotavirus, astrovirus and adenovirus are common pathogens that cause infection. The current methods for diagnosing these viruses mainly include immunological methods and PCR-based Nucleic acid detection methods, these methods often require cumbersome operating procedures, relatively expensive inst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2521/101
Inventor 叶辛方雪恩孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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