Multi-target gene parallel detection combined probe and application thereof

A combined probe and multi-target technology, used in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of multiple types of primers, high concentration, detection cross-interference, etc., and achieve strong exponential amplification ability, improved accuracy and specificity, and structurally stable effects

Active Publication Date: 2019-09-03
XI AN JIAOTONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] However, in the existing detection methods, due to the many types and high concentrations of primers involved in the multiplex amplification system, the parallel detection of multiple targets has serious cross-interference, and the resolution of target sequence recognition is insufficient.
Therefore, it remains a challenge to develop methods that can accurately identify single-nucleotide variants in multigene parallel testing

Method used

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  • Multi-target gene parallel detection combined probe and application thereof
  • Multi-target gene parallel detection combined probe and application thereof
  • Multi-target gene parallel detection combined probe and application thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0061] The multi-target gene parallel detection of the above-mentioned combination probe type B hepatitis B virus of embodiment 1

[0062] combine Figure 7 In the detection results shown, channels 1, 2, 3, and 4 correspond to the fluorescent signals of Tex, Hex, FAM, and Cy5, respectively. When hepatitis B exists in the system, FIP-Hex and BIP-Tex interact with the target S gene and C gene respectively, and channels 1 and 2 both generate strong fluorescent signals. The amplification product of LAMP is completely complementary to the cohesive end of the B-type specific hybridization structure probe (LF-FAM / LF Block BHQ2), which can produce a strong Toe-hold displacement, and channel 3 can collect strong FAM fluorescence . Simultaneously displacing the bound FAM fluorescent probe can be used as a loop primer to accelerate the amplification of the hepatitis B S gene. Due to the base difference between hepatitis B virus type B and type C hepatitis B virus genome, the ability o...

Embodiment 2

[0063] Example 2 The above combined probes can be used for parallel detection of multi-target genes of hepatitis B virus

[0064] combine Figure 8 In the detection results shown, channels 1, 2, 3, and 4 correspond to the fluorescent signals of Tex, Hex, FAM, and Cy5, respectively. When hepatitis B exists in the system, FIP-Hex and BIP-Tex interact with the target S gene and C gene respectively, and channels 1 and 2 both generate strong fluorescent signals. The amplification product of LAMP is completely complementary to the cohesive end of the C-type specific hybridization structure probe (LB-Cy5 / LB Block BHQ2), which can produce a strong Toe-hold displacement, and channel 4 can collect strong FAM fluorescence . Simultaneously displacing the bound Cy5 fluorescent probe can be used as a loop primer to accelerate the amplification of the hepatitis C S gene. Due to the base difference between hepatitis B virus and hepatitis B virus genome, the ability of the above-mentioned L...

Embodiment 3

[0065] Example 3 The above combined probes can be used for parallel detection and typing of multiple target genes of hepatitis B virus in clinical serum samples

[0066] 300 μL of serum samples were collected from 24 patients with hepatitis B, and the virus genome in the serum was extracted with a commercial kit. Through the effective integration of multi-channel information and mutual verification of data, high-quality data for target quantification and typing can be accurately obtained. The result is as Figure 9 As shown: samples No. 2 and No. 14 are hepatitis B virus, samples No. 1 and 11 are other subtypes of hepatitis B virus, and the rest are hepatitis B virus type C. The above results are consistent with the sequencing results.

[0067] In summary, the present invention constructs a multiplex LAMP amplification system, and the signal probe combination includes: a pair of stem-loop structure probes (FIP-Hex, BIP-Tex), a pair of hybridization structure probes (LF-FAM / LF...

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Abstract

The invention discloses a multi-target gene parallel detection combined probe and an application thereof. The probe comprises a pair of stem-loop structure probes and a pair of double-stranded hybridstructure probes; wherein the stem-loop structure probes are formed by connecting complementary sequences of 5' end of 5' oligonucleotide strand through a C18 spacer, the stem-loop structure probe with a C18 as a loop and carrying a 3' oligonucleotide protruding single strand; the double-stranded hybrid structure probe comprises a long strand of a 5'-terminally labeled fluorophore and a short strand of 3'-label quenching group, a full length of the short strand is partially complementary to the long strand to form a double-stranded complex. The designed multi-color fluorescence-labeled structural nucleic acid probe combination can be used for simultaneous detection of multi-target genes with sensitivity up to 1 copy / microliter; combined with dual regulation of strand displacement thermodynamics and polymerase kinetics, the recognition resolution reaches a mononucleotide level, and the accurate identification of mononucleotide variation in multi-gene parallel detection is realized.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and relates to the application of a multi-target gene parallel detection combination probe and a kit thereof. Background technique [0002] Multi-gene joint detection can simultaneously obtain multi-gene sequence, locus and abundance information, comprehensively obtain sample characteristics, and effectively improve the reliability and accuracy of analysis and detection, which is of great significance in the fields of biological research and disease diagnosis. Multiplex PCR amplification technology can use multiple pairs of specific primers to simultaneously amplify multiple target sequences in the same reaction system, and has been widely used in multiple gene joint detection. However, multiplex PCR technology needs to rely on a thermal cycle device that can precisely control the temperature, which is difficult to meet people's analysis needs for simple and convenient molecular diagnostic...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/119C12Q2537/1373C12Q2563/107
Inventor 赵永席赵越房晓星陈锋
Owner XI AN JIAOTONG UNIV
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