An Overlapping Loop-Mediated Isothermal Nucleic Acid Amplification Technique

A ring-mediated isothermal and nucleic acid technology, applied in the field of warm nucleic acid amplification technology, can solve the problems of difficult double gene or even multigene detection, large reaction interference, cumbersome and complicated system, etc., and achieve simple primer design method and high reaction efficiency , the effect of accurate response results

Active Publication Date: 2022-02-18
SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the large number of primers used for LAMP amplification, it is difficult to perform double-gene or even multi-gene detection in the same reaction system
At present, although there is a probe method that can perform multi-gene detection in the same reaction system, this method uses many primers, the system is relatively cumbersome and complicated, the design of primers is difficult, the reaction interference is relatively large, and the application prospect is not ideal

Method used

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  • An Overlapping Loop-Mediated Isothermal Nucleic Acid Amplification Technique
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  • An Overlapping Loop-Mediated Isothermal Nucleic Acid Amplification Technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Design overlapping primers and amplify MS2-SGIV-specific gene effect display

[0036] 1. Design primers to detect MS2-specific genes. The MS2 gene sequence is shown in SEQ ID NO.1, and the MS2 primers are designed as figure 1 As shown, the primer sequences are specifically shown in Table 1, and the reaction system is shown in Table 2.

[0037] Table 1 MS2 primer sequences for loop-mediated isothermal nucleic acid amplification

[0038]

[0039] Table 2 Reaction system

[0040]

[0041] The above reaction system was carried out in a constant temperature water bath at 65°C for 60 minutes, and nucleic acid electrophoresis was carried out. The results were as follows: figure 1 (1~4). figure 2 Among them, M is DL2000 DNA marker, 1 and 2 are MS2 reaction gel images, LAMP reacts normally, 3 and 4 are negative controls, only PUC19 plasmid DNA is added as negative control.

[0042] 2. Design primers to detect SGIV-specific genes. The SGIV gene sequence is...

Embodiment 2

[0066] Example 2: Design overlapping primers and amplify CCHFV-HCV specific gene effect display

[0067] 1. Design primers to detect CCHFV-specific genes. The CCHFV gene sequence is shown in SEQ ID NO.3, and the CCHFV primers are designed as follows Figure 5 As shown, the primer sequences are specifically shown in Table 11, and the reaction system is shown in Table 12.

[0068] Table 11 Loop-mediated isothermal nucleic acid amplification CCHFV primer sequences

[0069]

[0070] Table 12 Reaction system

[0071]

[0072] The above reaction system was carried out in a constant temperature water bath at 65°C for 60 minutes, and nucleic acid electrophoresis was carried out. The results were as follows: Image 6 (1~4). Image 6 Among them, M is DL2000 DNA marker, 1 and 2 are CCHFV reaction gel images, LAMP reacts normally, 3 and 4 are negative controls, only PUC19 plasmid DNA is added as negative control.

[0073] 2. Design primers to detect HCV specific genes. The HCV...

Embodiment 3

[0091] Example 3: Design of overlapping primers to amplify MS2-HCV-SGIV-specific gene effect display

[0092] 1. Design double-gene MS2-HCV and double-gene HCV-SGIV overlapping primers respectively according to step 3 of embodiment one and two, and design LAMP primers according to the principle of multigene design; specifically as Figure 9 shown. The primer sequences are specifically shown in Table 19, and the reaction system is shown in Table 20.

[0093] Table 19 Overlapping loop-mediated isothermal nucleic acid amplification MS2-HCV-SGIV primer sequences

[0094]

[0095] Table 20 Reaction system

[0096]

[0097] The above reaction system was carried out in a constant temperature water bath at 65°C for 60 minutes, and nucleic acid electrophoresis was carried out. The results were as follows: Figure 10 (1~4). Figure 10 Among them, M is DL2000 DNA marker, 1 and 2 are three-gene reaction gel images, LAMP reacts normally, 3 and 4 are negative controls, and only PU...

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Abstract

The invention belongs to the field of biological detection, and in particular relates to an overlapping loop-mediated isothermal nucleic acid amplification technology. The specific technical scheme includes: an amplification primer for a loop-mediated isothermal nucleic acid amplification technique, the amplification primer is an overlapping primer, and the overlapping primer includes a forward primer F and a reverse primer R, and the right side of the forward primer F is It is the normal PCR primer F for splicing the downstream DNA of the DNA sequence, the left side is the specific sequence with the upstream DNA sense sequence or complementary to the reverse primer R, the left side of the reverse primer R is the normal PCR primer R for splicing the upstream DNA of the DNA, and the right side is Contains a downstream DNA antisense sequence or a specific sequence complementary to the forward primer F. The present invention provides a new LAMP primer design and gene detection method, and proposes the concept and design idea of ​​"overlapping primer" for the first time. By designing specific overlapping primers, it helps multiple genes to realize overlapping loop-mediated isothermal nucleic acid amplification reaction , the primer design method is simple, the reaction result is accurate, and the reaction efficiency is high.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to an overlapping loop-mediated isothermal nucleic acid amplification technology. Background technique [0002] Loop-mediated isothermal nucleic acid amplification technology (loop-mediated isothermal amplification, LAMP) is a new constant temperature nucleic acid amplification method, specifically for the design of 4 specific primers for 6 regions of the target gene, using strand displacement DNA polymerase isothermal (around 65°C) for a period of time to complete the nucleic acid amplification reaction. Compared with conventional PCR, LAMP does not require thermal denaturation of the template, temperature cycling, electrophoresis, and ultraviolet observation, and has the characteristics of simplicity, rapidity, strong specificity, and high sensitivity. [0003] However, due to the large number of primers used for LAMP amplification, it is difficult to perform doubl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2545/113C12Q2565/125C12Q2537/1376
Inventor 胡学军
Owner SHANGHAI ZHONGQI BIOTECHNOLOGY CO LTD
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