Primer-probe combination for screening diseases, application of primer-probe combination, kit and detection method

Abstract

The invention discloses a primer-probe combination for simultaneously screening spinal muscular atrophy and primary immunodeficiency diseases, application of the primer-probe combination, a detectionkit and a detection method. The detection kit comprises primers and probes for screening and detecting a spinal muscular atrophy gene and primers and probes for screening and detecting genes (TREC andKREC) of the primary immunodeficiency diseases (advanced-case combined immunodeficiency disease and agammaglobulinemia). According to the primer-probe combination, the application of the primer-probecombination, the detection kit and the detection method, a multiple digital PCR detection system with high sensitivity, high accuracy, low cost and short cycle can be established, quantified detection on a copy number of a pathogenic gene of a hereditary disease, i.e., the spinal muscular atrophy (SMA), a copy number of an advanced-case combined immunodeficiency disease related marker TREC and acopy number of an agammaglobulinemia related marker KREC can be achieved simultaneously in one reaction, and thus, the above-mentioned three kinds of diseases are screened in one time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the field of nucleic acid detection, and more particularly, to a multiple digital PCR detection method and primers for simultaneously screening genetic diseases Spinal Muscular Atrophy (SMA) and primary immunodeficiency disease Probe combinations, detection reagents, and kit applications. Background technique [0002] Spinal muscular atrophy (SMA) is an inherited neuromuscular disease characterized by degeneration of motor neurons in the anterior horn of the spinal cord, resulting in severe, progressive, symmetrical muscle weakness and atrophy. People with SMA may be incapacitated and have difficulty performing basic life functions such as breathing and swallowing. Without treatment, most infants with the more severe form of the disease (SMAI) do not live to age two without respiratory intervention. According to statistics, the incidence rate of neonatal SMA is 1 / 6000~1 / 10000, and ...

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Problems solved by technology

[0009] 1. The multiple ligation-dependent probe amplification (MLPA) method is used to detect the SMN1 gene copy number. The experimental operation is complicated and the cycle is relatively long. The general experimental operation takes about 3 days. The reagents are expensive and it is not easy to promote clinically.

[0010] 2. Fluorescent quantitative PCR method to detect SMN1 gene copy number needs to rely on the detection results of normal control samples, and the calculation is more complicated, and it is impossible to distinguish the copy number changes of small changes

[0011] 3. The PCR-RFLP method to detect the SMN1 gene copy number can only detect the homozygous deletion, and cannot detect carriers containing 1 copy

[0012] 4. The fluorescent quantitative PCR method is used to detect the copy number of TREC and KREC. The real-time fluorescent quantitative amplification method mainly uses the curve of the standard quality control product to perform relative quantification on the copy number of the target sequence, and the accuracy and stability of the detection results are affected by the standard product. Larger, which eventually leads to higher false positives and false negatives

In addition, digital PCR methods are currently used to detect the copy number of SMN1 or TREC and KREC alone, but there is no technical solution that can simultaneously detect the above indicators and realize the simultaneous screening of three genetic diseases

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