Multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting multiple influenza viruses and application of multiple LAMP primer combination

A combination technology of influenza virus and primers, applied in the biological field, can solve problems such as false positives of primers, and achieve the effects of short reaction time, high sensitivity, and improved sensitivity

Active Publication Date: 2021-07-20
厦门健康工程与创新研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing multiple LAMP detection of influenza virus has technical problems that false positives are easily caused between primers, and the reaction speed and sensitivity need to be further improved

Method used

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  • Multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting multiple influenza viruses and application of multiple LAMP primer combination
  • Multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting multiple influenza viruses and application of multiple LAMP primer combination
  • Multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting multiple influenza viruses and application of multiple LAMP primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This embodiment provides a combination of multiple LAMP primers for detecting multiple influenza viruses, refer to figure 1 As shown, in this embodiment, a pair of primers F4 and B4 are added around the traditional F3 and B3 to improve the reaction speed and sensitivity. The difficulty of adding a pair of primers mainly lies in the primer design, and too many primers can easily cause primer dimers. The primer sequences of multiple LAMP primer combinations obtained through final design and screening in this embodiment are shown in Table 1.

[0058] The primer sequence of the multiple LAMP primer combination of table 1 embodiment 1

[0059]

[0060]

[0061] Note: M1, M2, and M3 are prefixes added for the convenience of distinguishing the first primer set, the second primer set and the third primer set, without changing the type of primers.

[0062] The primers of each primer set in Table 1 were artificially synthesized for future use. In the first primer set, th...

Embodiment 2

[0065] This embodiment provides a multiplex LAMP detection method, which utilizes the primer combination of Example 1 for LAMP detection or first designs and synthesizes the multiplex LAMP primer combination of Example 1.

[0066] After obtaining the multiple LAMP primer combination of Example 1, the multiple LAMP detection method for multiple influenza virus detection specifically includes the following steps:

[0067] The multiple LAMP primer combination is made into primer MIX, and in the primer MIX, the concentration of FIP and BIP is 1.6 μM, the concentration of B3 and F3 is 0.2 μM, the concentration of LF and LB is 0.4 μM, and the concentration of F4 and B4 is 0.3 μM; Where μM is the abbreviation of μmol / L commonly used in the industry.

[0068] S2. Prepare the reaction system to carry out the loop-mediated isothermal amplification reaction. In this embodiment, the detection of the sample to be tested is used as a test group, and the reaction system includes the DNA temp...

Embodiment 3

[0081] This embodiment provides a multiple LAMP detection method, which adds a sample collection and mixed detection step on the basis of the steps in Embodiment 2. This step specifically includes: collecting samples to be tested, and mixing multiple samples to be tested for mixed testing. The number of samples to be tested is 5 or 10.

[0082] Mixed detection is a common way to reduce the number of detections, and it is often used in conjunction with PCR (Polymerase Chain Reaction, polymerase chain reaction) detection. However, the disadvantage of mixed detection is: mixing the samples will reduce the concentration of the sample to be tested, which may be lower than the lower limit of PCR detection; resulting in unsatisfactory mixed detection results; but the sensitivity (lower limit of detection) of LAMP is 100 times higher than that of conventional PCR, so mixed detection is of great importance. The effect of LAMP test results is much smaller than that of PCR test.

[008...

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Abstract

The invention discloses a multiple LAMP (loop-mediated isothermal amplification) primer combination for detecting multiple influenza viruses and application of the multiple LAMP primer combination, the traditional 6-region 2-pair primers are improved into 8-region 4-pair primers, and F4 and B4 primers are added, so that the detection time is shortened, the detection sensitivity is improved, and false positive cannot be caused among the primers. The multiple LAMP primer combination for detecting multiple influenza viruses provided by the invention can be used for detecting whether influenza A H1N1, influenza A H3N2 or influenza B is infected, is efficient and rapid, and is beneficial to popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination of multiple LAMP primers for detecting various influenza viruses and an application thereof. Background technique [0002] Influenza virus is a representative species of Orthomyxoviridae (Orthomyxoviridae), referred to as influenza virus, which is divided into influenza A virus (such as influenza A H1N1 virus, influenza A H3N2 virus, etc.), influenza B virus There are three types of influenza virus and type C, among which type A is the most likely to cause epidemics, followed by type B, and type C rarely causes epidemics. [0003] Loop-mediated isothermal amplification technology (1oop-mediated isothermal amplification) is a simple, rapid, specific, and low-cost nucleic acid amplification technology that can be completed in the next step under isothermal conditions. This method was established by Notomi et al. in 2000 , has been widely used in pathogenic mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2537/143Y02A50/30
Inventor 蔡文凯雷洋陈凯君白佳委陈伟川曾晓昕石岩汪大明
Owner 厦门健康工程与创新研究院
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