Amplification composite for detecting microdeletion of Y-chromosome and detection kit

Abstract

The invention relates to amplification composite for detecting the microdeletion of Y-chromosome and a detection kit, belonging to the field of biotechnical detection. The amplification composite for detecting the microdeletion of Y-chromosome can be amplified to as many as 30 sites related to the microdeletion detection of the Y-chromosome through one reaction. The detection kit detects the microdeletion of the Y-chromosome through the quantitative fluorescent PCR (Polymerase Chain Reaction) method by using the amplification composite. The microdeletion abnormality of the Y-chromosome is determined according to the existence of an amplification product and the quantity of the amplification product. A large quantity of sites enables the detection result to be more convincible and can provide much more and more detailed information for determining the deletion type, and the quantitative detection of partial deletion and repetition can be realized. The detection kit is easier and more convenient to operate, only one PCR amplification and one sequencer detection reaction are needed to complete the detection of one sample, the whole process needs 4-5 h, and the operation intensity and the detection time are greatly reduced.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to an amplification composition for detection of Y chromosome microdeletion, the detection method and a detection kit. Background technique [0002] About 15% of couples of childbearing age have fertility disorders, of which about half are caused by male factors. Among the known genetic factors that lead to male infertility, the two most common are Y chromosome microdeletion and Klinefelter syndrome, with an incidence rate of 10-15% in azoospermia or oligospermia patients. [0003] The Y chromosome has a large number of repetitive sequences and a palindromic structure. While maintaining the evolutionary stability of the Y chromosome, these structures also make the genes inside the palindromic structure easy to lose, resulting in infertility. The three spermatogenesis-affecting regions with the highest deletion rates were named AZFa, AZFb, and AZFc, and deletions in any of the...

AI Technical Summary

Problems solved by technology

[0008] ①, multiple PCR amplification reactions are required to detect one sample, which is costly and intensive to operate

[0009] Due to the use of agarose electrophoresis detection, the resolution and sensitivity of the method are limited, and it is difficult to simultaneously amplify a larger number of sites in each amplification reaction. Currently, products on the market and related patents only amplify 4-5 sites in each amplification reaction site

Therefore, even if only the EAA / EMQN standard sites are detected, 2 amplification reactions are required, and more amplification reactions are required as the number of detection sites increases (for example, the sub-energy kit requires 4 reactions, and the Promega kit Detecting a sample requires 5 amplification reactions and 5 detections), which greatly increases the operating intensity and detection cost, and the workload is very large for large-scale sample screening

[0010] ②, the amplified product is detected by agarose electrophoresis, the detection efficiency is low, the sensitivity is low, and the error rate is high

[0011] Agarose electrophoresis requires a lot of manual operations. It takes 1-2 hours from gel preparation to sample application, electrophoresis and signal reading, which is not convenient for the detection of a large number of samples; moreover, the detection sensitivity of agarose electrophoresis is low, and the bands are not easy when the product amount is small. Discrimination, it is difficult to distinguish between weak positives and negatives, and it is easy to cause false positives or false negatives; if the electrophoresis conditions are unstable or improperly operated, it is easy to cause sample confusion and wrong judgments

[0012] ③, unable to detect partial deletions and duplications

[0014] However, since the AZFb / c region sequence with high partial deletion and duplication is composed of a highly consistent palindromic structure, it is difficult to select a single-copy specific site for detection; at the same time, because multiple qualitative PCR technology can only detect the presence or absence of amplified products. None, cannot be quantitatively detected to distinguish between partial deletions and duplications of repetitive DNA fragments on chromosomes

Therefore, multiple qualitative PCR technology is difficult to detect partial deletions and duplications, and will misjudge partial deletions and duplications as normal, affecting subsequent diagnosis and treatment or related treatments

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