Development Of Deletional α-thalassemia Gene Copy Number Quantification Test Kit

Background and ObjectivesThalassemia (referred to as thalassemia) is due to globin gene defects, resulting globin chain synthesis reduced or absent, so that the formation of hemoglobin a-chain/non-a-chain imbalance, caused by a group of serious threat to human healthy death, disabling inherited blood disease occurs mainly in Mediterranean countries, Southeast Asia, Africa and southern China, a conservative estimate of thalassemia gene carriers around the world nearly200million people, including southern China, Guangxi, Guangdong, Hainan and other provinces. Thalassemia is major consist of a-thalassemia and β-thalassemia. In Guangxi, Guangdong, a-thalassemia gene carrier rate is as high as17.55%and8.53%, respectively. The disease is currently a lack of effective treatment, the application of appropriate analytical techniques through the crowd to stop screening and prenatal diagnosis of children with severe birth is widely recognized as the preferred preventive measures.Alpha-thalassemia are autosomal recessive inherited disease, the molecular basis of human congenital genetic defect is located on the16th chromosome16p13.3locus ends on a-globin gene cluster. Resulting in decreased synthesis of a-chain gene defect major (>95%) is the a-globin gene deletion, the missing number is a-thalassemia clinical presentation and classification basis. In southern China, three common α-thalassemia deletion genotypes are--SEA/αα (Southeast Asian type),-α3.7/αα (Right deletional type) and-α4.2/αα (left deletional type).At present, for a-thalassemia screening only stay in hematology, hematology obvious that only individuals phenotype will be further molecular diagnostics, which causes most of the slient carriers (such as-α3.7/αα and-α4.2/αα) missed, so for a-thalassemia screening in urgent need of a simple, rapid genotyping screening tool. However, for a-thalassemia molecular diagnostics, Southern Blot hybridization is the gold standard, high accuracy, but complicated operation, a large amount of samples, time consuming, and not suitable for the detection of small throughput routine testing; Current conventional technology is gap-PCR technique, known only qualitatively detect the missing type, heavy workload, testing flux is small, and there is a lack of other types of pollution leading to false-negative and false-positive PCR, it still can not meet the current large-scale population screening and conventional molecular diagnostics needs.Our research team invented a method (Patent No.:ZL201110081598.8) a direct quantification of gene copy number with rapid diagnostic deletional a-thalassemia, the method is the use of TaqMan real-time quantitative PCR fluorescence quantitative analysis techniques directly ζ, α1and relative copy number of a2to diagnose the existence of missing, effectively avoiding the type of missing genetic heterogeneity caused by exogenous contamination and false-negative result in false positives, and easy to automate and standardize and improve the detection throughput and detection efficiency, suitable deletional a-thalassemia screening of large populations and conventional molecular diagnostics.This study is based on the patent technology, through extract genomic DNA and establish the PCR amplification detection system to developing a kit that can be used for routine clinical applications, to meet the current needs of clinical testing.Programs and methodsA molecular diagnostic method involves sample source, sample preparation, sample testing, result analysis step, only do quality control at each step in order to ensure accurate and reliable results, therefore, based on this complete diagnostic process, this study implemented the following specific research programs and methods. 1. Sample collection, transportation and preservation. The effects of different anticoagulants on PCR amplification is different, for example, heparin blood samples after DNA extraction by PCR amplification efficiency will affect. Degree will also affect the quality of fresh blood for DNA extraction. Therefore, blood samples were used in this study were asked to EDTA anticoagulated whole blood, stored at4℃refrigerator no more than7days; At-20±5℃storage period of six months; To long-term preservation, should be stored at-80±5℃; Specimens avoid repeated freezing and thawing.2. Whole blood genomic DNA extraction. Human genomic DNA samples for PCR amplification of concentration and purity have a greater impact, such as human genomic DNA sample concentration is too low (below detection limit) or contain PCR inhibitors will lead to PCR amplification affected. Therefore, this study from the erythrocyte lysate, denaturant, digestion time, DNA lysate extract and other aspects of DNA extraction methods Development of a comprehensive, DNA extraction method developed to meet the requirements of real-time PCR. UV spectrophotometer to monitor the use of DNA samples for concentration and purity, the purity of better OD260nm/OD280nm ratio of human genomic DNA samples should be between1.6to2.0, while the concentration in20ng/ul that can satisfy the above PCR amplification kit increasing demand (the amount of DNA using this kit is5ul).3. Raw material quality testing. Raw material quality standards are basic requirements for a successful experiment, so the quality testing of raw materials is particularly important. The main raw materials, include proteinase K, guanidine hydrochloride, primers, probes and polymerase. Proteinase K should be white floc or powder; guanidine hydrochloride is a white crystalline powder should, AR; primers and probes should be used to determine the concentration of UV spectrophotometer, using polyacrylamide gel electrophoresis purity identification, concentration of not less than synthetic concentration, electrophoresis results should be in the correct position of a single band, which means that the purity of qualified; polymerase should be colorless transparent viscous liquid at-20±5℃not freeze. 4. Kit preparation and verification. The kit includes whole blood genomic DNA extraction kit and PCR assay. Genomic DNA extraction reagents include erythrocyte lysis buffer, DNA extraction solution A (buffer), DNA extraction solution B (denaturing agent), DNA extracts were C (protease), DNA extract D, DNA lysate. PCR detection kit comprises a PCR reaction solution A (buffer, enzyme), and the PCR reaction solution B (primers, probes). Need all the liquid preparation prepared in accordance with the requirements, according to the prepared liquid corresponding requirements after sterilization or directly store to store. The prepared whole blood genomic DNA extraction kit (column formulation) combines quality inspection kit other components to the normal genotype calibrators1,2, positive reference product P1, P6, P8and negative control of sample, the applicable instrument, strictly operate according to kit instructions. The preparation of a good HBA PCR reaction solution A combination of good preparation HBA PCR reaction solution B formulated into a PCR reaction tube to the normal genotype calibrators1,2, the minimum detectable amount of reference material L1, L2and negative control samples in the applicable instrument, strictly operate according to kit instructions. After the completion of reagent preparation, validate full reagents. Full kit finished by PCR assay verification program for testing.5. The kit of clinical trials. By deletional a-thalassemia gene copy number quantification and detection kit provided by Sun Yat-Da An Gene Co., Ltd."a-thalassemia gene detection kit (gap-PCR method)", while the Guangxi Zhuang Autonomous Region Maternity and child care center, Liuzhou Maternity and child care center and Guangzhou Women and Children medical Center collected and extracted human genomic DNA samples obtained were blinded testing. With gap-PCR method for the control, inspection sensitivity of PCR-fluorescent probe, specificity, in line with rate detection performance indicators. The results do not match the specimen using MRC-Holland’s Dutch SALSA (?) reagents for MLPA (?) reactions (P140HBA probemix, CY5mark, BeckMan GeXP detected) review test.Results According to the research objectives and research strategy, through the implementation of specific research programs and methods, the results obtained are as follows:1. Sample collection, transportation and preservation. Blood samples used in this study are in line with the requirements of the experimental method, which are EDTA anticoagulated whole blood, stored at4℃refrigerator no more than7days.2. Whole blood genomic DNA extraction. Developed a whole blood genomic DNA extraction methods of the quantitative PCR kit required to meet. The series of optimization and adjustment, DNA extraction methods required to determine the whole blood200ul, with lysate as NH4C1, trizol as denaturing agent guanidine hydrochloride and pure water as extracting agent is dissolved as a DNA solution,56℃warm bath for half an hour can be extracted, the extraction process takes50minutes. Under such conditions, the extracted DNA concentrations between20～50ng/ul, OD260nm/OD280nm ratio between1.6to2.0; description of this whole blood genomic DNA extraction methods in line with the real-time PCR kit requirements.3. Quality testing kit materials. Proteinase K is white floc or powder; guanidine hydrochloride is a white crystalline powder, AR; primers and probes using UV spectrophotometer concentration, concentration of not lower than the concentration of the synthesis, using polyacrylamide gel electrophoresis purity identification electrophoresis results in the correct position of a single band, which means that the purity of eligibility; polymerase should be colorless transparent viscous liquid at-20±5℃not freeze.4. Kit preparation and verification. Each kit components are formulated in accordance with the requirements, according to the prepared liquid corresponding requirements after sterilization or directly store to store. Good preparation reagents validated using quality control materials, quantitative test results all right.5. The kit of clinical trials. Accordance with the test design requirements, to gap-PCR method as a control, were detected1,449cases of human genomic DNA samples, of which685cases and Child Health Hospital of Guangxi, Liuzhou MCH303cases, Guangzhou Women and Children Medical Center461cases. Use gap-PCR method detected positive (deletional a-thalassemia)855cases, negative (excluding deletional a-thalassemia)530cases; using fluorescence quantitative detection kit-positive (deletional a-thalassemia)847cases negative (excluding deletional a-thalassemia)538cases. The kit is calculated by fluorescence quantitative statistical methods to detect deletional a-thalassemia mutation positive coincidence rate (99.67%), negative coincidence rate (99.81%), thick consistency (99.72%) and Kappa values (0.994).ConclusionAfter a control sample sources, development of whole blood genomic DNA extraction methods, quality testing kit for each component of raw materials, formulation and validation of clinical trial kits and kits of components, the paper developed deletional a-Mediterranean anemia gene copy number quantification test kit reagents meet the requirements of clinical testing, clinical assessment of qualified, suitable for large-scale population screening deletional a-thalassemia and routine diagnostics.