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Detection method of adult type polycystic kidney

A detection method, polycystic kidney technology, applied in the field of genetic disease gene detection, can solve the problems of not providing detection methods, and achieve the effect of facilitating standardized batch sample detection, ensuring reliability, and eliminating the interference of pseudogenes

Inactive Publication Date: 2017-09-26
杭州博圣医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention involves six gene detection regions capable of detecting autosomal dominant polycystic kidney disease and autosomal recessive polycystic kidney disease, but does not provide a detailed and rapid detection method

Method used

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  • Detection method of adult type polycystic kidney
  • Detection method of adult type polycystic kidney
  • Detection method of adult type polycystic kidney

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Next Generation Sequencing Detection

[0052] 1. Multiplex PCR

[0053] Through the verification of multiple PCR enzymes, the present invention selects the enzyme KOD-Multi&Epi with the best performance in terms of fidelity, amplification efficiency, and amplification balance of fragments of different lengths. The primers were designed independently and referenced in the literature, and the specific sequences are shown in Table 1.

[0054] Table 1 Primer information

[0055]

[0056]

[0057] 1.1 Reaction system

[0058]

[0059] 1.2 Reaction conditions:

[0060] PKD1

[0061]

[0062] PKD2:

[0063]

[0064] 1.3 PCR product electrophoresis quality inspection

[0065] 2. PCR product recovery

[0066] Ampure XP beads were used for purification in this experiment.

[0067] (1). Take a 1.5ml centrifuge tube and mix the PCR products of the above 7 groups at a ratio of 1:1. That is 7X 10ul, about 63ul after mixing.

[0068] (2). Add 0.1× volume fract...

Embodiment 2

[0201] Generation Sequencing Validation

[0202] After the results of the next-generation sequencing analysis are obtained, the mutation points detected by the next-generation sequencing need to be verified by first-generation sequencing. Long-fragment amplification is performed on the fragment where the mutation point detected by the next-generation sequencing is performed, and verification primers are designed for the mutation point. After the amplification is completed, the verification primer is used to perform first-generation sequencing verification on the mutation point detected by the second-generation sequencing.

[0203] The sequencing results are bidirectionally checked and consistent, and all have uniform detection results for the mutation point (such as Figure 8 ). The disease-causing mutation sites verified by first-generation sequencing can be identified as the detection results.

[0204] 1 PCR

[0205] After the results of the next-generation sequencing ana...

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PUM

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Abstract

The invention relates to a detection method of adult type polycystic kidney. The treatment mutation of adult type polycystic kidney disease-causing genes PKD1 and PKD2 is detected and identified by adopting a long fragment PCR (Polymerase Chain Reaction) capturing, second generation sequencing and first generation identification. Compared with an existing probe capturing technology, true and false genes can be distinguished, real genes are specifically captured and interference caused by the false genes is reduced; the ratio of all components is optimized and the consistency of capturing is ensured; the conditions that the uniformity of data is poor and the data quantity of partial segments is not enough are avoided; an optimized database building scheme has good stability and automatic database building is convenient to carry out; after an existing analysis method is tested, a detection result can be processed very well and a correct result is found.

Description

technical field [0001] The invention belongs to the technical field of genetic disease gene detection and relates to a detection method for adult polycystic kidney disease. Background technique [0002] Polycystic kidney disease is a common hereditary kidney disease. It is mainly manifested by multiple cysts of different sizes in both kidneys. The cysts progressively enlarge and occupy space, squeeze normal nephrons, destroy the structure and function of the kidney, and eventually lead to End-stage renal disease. The clinical manifestations of the disease are multiple renal cysts, progressive renal failure, persistent hypertension, and urinary tract infection. It can also be combined with other organ cysts (liver cysts, pancreatic cysts, ovarian cysts, etc.), cerebrovascular malformations, Aneurysm, heart valve disease, portal hypertension and other complications. Due to the lack of effective treatment methods, most patients develop end-stage renal failure and have to unde...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/113C12Q2537/143
Inventor 俞晓敏杨丽萍侯敏罗旌万饶欢
Owner 杭州博圣医学检验实验室有限公司
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