Method for increasing compatibility of multi-PCR (Polymerase Chain Reaction) primer

A compatible and multiple technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long amplification time and difficult design of multiple PCR amplification primers, so as to reduce the amplification background and reduce the The difficulty of primer design and the effect of increasing compatibility

Inactive Publication Date: 2019-03-19
SHANDONG ACV BIOTECH CO LTD
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] In order to make up for the deficiencies of the prior art and solve the problems of the prior art that the design of multiplex PCR amplification primers is difficult, easily lead to amplification skewness, and long amplification time, the present invention provides a method for increasing the compatibility of multiplex PCR primers (Multiple Primer Compatible PCR, Mpc-PCR)

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  • Method for increasing compatibility of multi-PCR (Polymerase Chain Reaction) primer
  • Method for increasing compatibility of multi-PCR (Polymerase Chain Reaction) primer
  • Method for increasing compatibility of multi-PCR (Polymerase Chain Reaction) primer

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Embodiment 1

[0061] In this embodiment, multiplex real-time fluorescent quantitative PCR technology platform is used for detection. Considering the channel limitation of the multiplex real-time fluorescent quantitative PCR technology platform itself, all primers are used in this embodiment for multiplex amplification, and only three indicators are selected for detection during detection.

[0062] With the No. 001 / 002 / 003 sample described in Table 4 as a template, a negative control (using DEPC water as a template) was added at the same time, and the specific primers and universal primers for all pathogens described in Table 2 were used as amplification primers. The amplification method provided by Mpc-PCR was invented for multiple amplification, and the probes of EV-U, EV71, and CA16 were used as detection probes (wherein the 5' of the EV-U detection probe was labeled with FAM fluorescence, and the 3' end was subjected to BHQ -1 fluorescent labeling; EV71 detection probe 5' is HEX fluoresc...

Embodiment 2

[0086] With No. 002 / 005 / 006 sample RNA described in Table 4 as a template, a negative control (using DEPC water as a template) was added simultaneously, and the specific primers and universal primers of all pathogens described in Table 2 were used as amplification primers for multiple amplification. In addition, the probes of CA6, CA10, and CA16 in Table 2 are used as detection probes (the 5' of the CA6 detection probe is fluorescently labeled with FAM, and the 3' end is fluorescently labeled with BHQ-1; the 5' of the detection probe of CA10 is fluorescently labeled with HEX labeling, BHQ-1 fluorescent labeling at the 3' end; Cy5 fluorescent labeling at the 5' end of the CA16 detection probe, and BHQ-2 fluorescent labeling at the 3' end), the multiplex fluorescent quantitative PCR technology platform was used as the detection platform for detection.

[0087] The present embodiment is carried out according to the following steps:

[0088] 1. The first step of PCR amplification ...

Embodiment 3

[0105] In order to test the sensitivity of this detection method, in this embodiment, the RNA of sample No. 001 / 002 / 003 / 004 / 005 / 006 described in Table 4 was used as a template, and a negative control (using DEPC water as a template) was added for detection. For each sample RNA, prepare five gradients: 100, 10 -1 、10 -2 、10 -3 、10 -4 . In another set of comparative experiments, the ratio of universal primer Rg:Fg was increased to observe the change of its detection sensitivity.

[0106] The specific primers and universal primers of all pathogens described in Table 2 were used as amplification primers for multiple amplification (wherein the reverse universal primer Rg was synthesized and the 5' end was labeled with biotin), and the specific primers of all pathogens described in Table 2 were used. The sex probe is the detection probe, and the liquid chip technology platform is used as the detection platform for detection.

[0107] The principle of liquid-phase chip technolog...

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Abstract

The invention discloses a method for increasing the compatibility of a multi-PCR (Polymerase Chain Reaction) primer and belongs to the field of biological detection. The method comprises the followingsteps: designing a pair of specific primers Fs and Rs and common primers Fg and Rg aiming at each target gene; carrying out enrichment and amplification on specific primers Fg+Fs and Rg+Rs with common primer labels; then carrying out index amplification by adopting the common primer labels Fg and Rg; and finally, detecting by adopting a probe hybridization or non-probe hybridization manner according to a detection platform. According to the method provided by the invention, each reaction component concentration, such as a primer concentration and an Mg<2+> concentration, in each step of PCR amplification is controlled to control reaction conditions of the PCR amplification, such as annealing temperature and annealing time, so that specific and sensitive amplification of multi-target genesis realized. The method has strong specificity and can ensure that the amplification efficiency of different pathogen nucleic acids is similar, and the deviation of the traditional multi-PCR amplification is avoided; and denaturalization does not need to be carried out to realize melting in a detection process.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for increasing the compatibility of multiple PCR primers. Background technique [0002] The molecular diagnostics market is a technological extension based solely on PCR (polymerase chain reaction), a Nobel-winning technology that has made revolutionary contributions to the field of biodiagnostics. PCR is a molecular biology technique used to amplify specific DNA fragments, which can be regarded as special DNA replication outside the body. The biggest feature of PCR is that it can greatly increase a small amount of DNA. However, in the application of this revolutionary diagnostic technology in clinical testing, most molecular diagnostic products based on PCR technology can only use a pair of primers to amplify one DNA target, that is to say: one kit can only detect one DNA target. index. This makes the development and application of PCR detection technology into a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q2537/143
Inventor 李艳艳张通靖相密
Owner SHANDONG ACV BIOTECH CO LTD
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