Reverse Dot Blot Assay Research Articles

Establishment and application of a reverse dot blot assay for 13 mutations of hearing-loss genes in primary hospitals in China.

Hearing loss is a common sensorineural dysfunction with a high incidence in China. Although genetic factors are important causes of hearing loss, hearing-related gene detection has not been widely adopted in China. Establishing a rapid and efficient method to simultaneously detect hotspot hearing loss gene mutations. A reverse dot blot assay combined with a flow-through hybridization technique was developed for the simultaneous detection of 13 hotspot mutations of 4 hearing loss-related genes including GJB2, GJB3, SLC26A4, and the mitochondrial gene MT-RNR1. This method involved PCR amplification systems and a hybridization platform. The technique can detect 13 hotspot mutations of 4 hearing loss-related genes. And a total of 213 blood samples were used to evaluate the availability of this method. Our reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify hotspot mutations of 4 hearing loss-related genes in a Chinese population.

Spontaneous miscarriage driven by maternal genetic mutation at position of PAI-1-844G/A: shed light on a race-specific genetic polymorphism

ObjectiveAssociation between a genetic polymorphism and disease, either positively or negatively, within a population may not necessarily predict association in other race-ethnic populations. The aim of this study was to genotype well recognized thrombophilia associated polymorphisms as common risk factors for miscarriage and investigate their benefit to use as risk factors in southwest region of Iran females (Khuzestan) in the Arabs ethnic minority group with spontaneous miscarriage. We developed a Reverse Dot Blot Assay for the genotyping of four polymorphisms.ResultsThere were significant differences in the genotype distribution and allelic frequencies of the MTHFR 1298 A > C, MTHFR 677 C > T, Factor V Leiden 1691 G > A, PAI-1-844G > A polymorphisms between the case and control groups. The MTHFR 1298 A > C, MTHFR 677 C > T and Factor V Leiden 1691 G > A polymorphisms were significantly associated with spontaneous miscarriage risk. Unlike some other race-ethnic populations, PAI-1-844G > A polymorphism was associated with risk of developing unplanned miscarriage in Iranian Arabs ethnic minority group females.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

Analysis of genotype-phenotype correlation in patients with α-thalassemia from Fujian province, Southeastern China.

BackgroundThere is a high carrying rate of α‐thalassemia in Fujian province. However, there are few large‐scale studies on the correlation between genotype and phenotype in Fujian province. The purpose of this study was to analyze the phenotype and genotype in a cohort of 2923 patients with α‐thalassemia in Fujian province, so as to provide reference data for screening and diagnosis of α‐thalassemia in Fujian province.MethodsThe genotype of α‐thalassemia was detected by PCR reverse dot blot assay, gap‐PCR, single PCR, nested PCR, and sequencing. Clinical and hematological indices of 2923 patients were collected, and the correlation between genotype and phenotype was analyzed.ResultsAmong 10,350 patients, 2923 cases were found with α‐thalassemia, with a detection rate of 28.24%. Among them, ‐‐SEA/αα was the most common genotype, accounting for 64.80%. In addition, rare α‐thalassemia genotypes were detected in Fujian province, including ‐‐THAI/αα (0.41%), HKαα/‐‐SEA (0.03%), and the novel α‐thalassemia gene mutation CD5 (GCC>ACC) (HGVS named HBA1: c.16G>A) (0.03%). Patients with deletional genotypes of α‐thalassemia were found to have higher RBC and lower Hb, MCV, MCH, and HbA2 than patients with non‐deletional genotypes of α‐thalassemia (p < 0.05).ConclusionThe clinical phenotype of α‐thalassemia is influenced by molecular mechanisms. HBA1: c.16G>A mutation is a novel mutation that was first reported in Fujian province, which enriches the human hemoglobin mutation spectrum.

Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Clinical validation of a single-tube PCR and reverse dot blot assay for detection of common α-thalassaemia and β-thalassaemia in Chinese

ObjectiveTo evaluate a novel reverse dot blot assay for the simultaneous detection six types of common α-thalassaemia alleles (three deletional and three common non-deletional mutations) and 19 types of common β-thalassaemia alleles in a Chinese population.MethodsGenomic DNA samples were collected from three hospitals in southern China. The novel thalassaemia gene assay involved one multiplex polymerase chain reaction amplification system and one round of hybridization. Each of the clinically validated DNA samples was re-tested using the new multiplex polymerase chain reaction/reverse dot blot assay II (M-PCR/RDB II) assay in a double-blind manner.ResultsA total of 1060 unrelated study participants, including 829 patients with thalassaemia and 231 healthy control subjects, were analysed. The whole PCR and RDB procedures were completed in 260 min. All the samples, including heterozygous thalassaemia, homozygous thalassaemia and compound heterozygous thalassaemia, were correctly genotyped, yielding 100% concordance with the reference assays. HKαα/--SEA and HKαα/−α4.2, which were not included in the detection panel, yielded a contradictory result with this new assay.ConclusionThe novel M-PCR/RDB II assay was simple, rapid and accurate, suggesting that it could be used for the genetic screening and clinical diagnosis of common α-thalassaemia and β-thalassaemia variants in Chinese populations.

Genotyping of Malaysian G6PD-deficient neonates by reverse dot blot flow-through hybridisation.

G6PD deficiency is the commonest enzyme deficiency found in humans. Current diagnostic methods lack sensitivity to detect all cases of G6PD deficiency. We evaluated the reverse dot blot flow-through hybridisation assay designed to detect simultaneously multiple known G6PD mutations in a group of Malaysian neonates. Archival DNA samples from 141 G6PD-deficient neonates were subjected to reverse dot blot flow-through hybridisation assay using the GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong) and DNA sequencing. The method involved PCR amplification of 5 G6PD exons using biotinylated primers, hybridisation of amplicons to a membrane containing oligoprobes designed for G6PD mutations known to occur in the Malaysian population and colour detection by enzyme immunoassay. The assay detected 13 of the 14 G6PD mutations and genotyped 133 (94.3%) out of 141 (102 males, 39 females) cases. Among the 39 female G6PD-deficient neonates, there were 7 homozygous and 6 compound heterozygous cases. The commonest alleles were G6PD Viangchan 871G > A (21%) and G6PD Mahidol 487G > A(20%) followed by G6PD Mediterranean 563C > T, (14%), G6PD Vanua Lava 383T > C (12%), G6PD Canton 1376G > T (10%), G6PD Orissa 131C > G (6.3%) G6PD Coimbra 592C > T (5.6%) plus 6 other mutations. DNA sequencing of remaining cases revealed 6 cases of intron 11 nt 93C > T not previously reported in Malaysia and two novel mutations, one case each of nt 1361G > T and nt 1030G > A. We found the reverse dot blot assay easy to perform, rapid, accurate and reproducible, potentially becoming an improved diagnostic test for G6PD deficiency.

Values of Hematological Indicators in the Screening of α-Thalassemia in Fujian Area of China

To analyze the genotypes and the hematological phenotypic characteristics of α-thalassemia in different areas of Fujian and to evaluate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (Hb), RBC distribution width/red blood cell (RDW/RBC) for screening α-thalassemia in this area. The Gap-PCR assay was applied for detecting 3 common deletional mutations of patients with α-thalassemia, and the reverse dot-blot (RDB) assay was adopted to detect the foci of 3 common non-deletional gene mutations.Then,the hematological parameters of individuals with α-thalassemia were analyzed. Finally, the optimal cut-off value in hematological indexes for screening α-thalassemia were determined by the ROC curve. Altogether 16 types of gene mutations were found in 772 patients with α-thalassemia. Among them, the -SEA/αα deletion mutation was the most common which was observed in 521 cases(67.49%). Compared with the control group, the differences in MCV, MCH, and Hb were statistically significant between the patients of the same sex but no same type. In male groups, the RDW/RBC ratio was statistically significant in individuals of light type and HbH disease as compared with the healthy control group. But in female groups, the statistical different of RDW/RBC ratio was found between only HbH disease group and control group. MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L, with the highest specificity and the highest sensitivity, were the best cut-off points for screening α-thalassemia in the laboratory. Due to the difference of regional heterogeneity and hospital equipment environment, the different laboratories need to establish cut-off value for screening α-thalassemia suitable for its local region. In future, our laboratory can use MCV<81.25 fl, MCH<27.30 pg, Hb(male)<128.5 g/L, and Hb(female) <123.5 g/L for value for clinical screening, of α-thalassemia.

Community-based prevalence versus hospital-based incidence of genital Human Papillomavirus infection in Central Vietnam.

This study aims to determine the genital HPV prevalence in reproductive-age women in Thua Thien Hue Province and comparison with HPV incidence in Hue University Hospital, Vietnam. Cross-sectional study on 1,034 women of reproductive age from 11 communes/wards of three districts representing three different geographic areas of Thua Thien Hue Province, Vietnam. The hospital-based group included 102 women with cervicitis and/or abnormal Pap smear result coming to Hue University Hospital. Extracting DNA from cervical samples, performing the real-time PCR for detecting HPV and the reverse dot-blot assay for HPV typing in HPV positive cases. In community, HPV prevalence was 0.9%. Mean-age of HPV positive group was 37.9 ± 6.2 years. The detected low-risk types were 6 and 11; high-risk types include 16, 18, 33, 45, 52, and 58. Single-type infection was found in 66.7% of cases. In hospital-based group, 41.2% of women have been infected with HPV, 6 different HPV types were detected. HPV18 was the most frequent high-risk type (33.3%), followed by HPV16 (15.1%); HPV6 was the most frequent among low-risk HPV types (31.2%). Single-type infection rate was 33,3%; 2 and 3 types co-infections were 28,6% and 38.1%, respectively. Routine screening of high-risk HPV infection in women with symptomatic gynecologic infection and/or abnormal Pap smear appears to be benefit in early detection and prevention of cervical cancer, due to the high incidence of HPV infection.

Analysis of genotype and hematological phenotype of 14 patients with coinheritance HKαα and South-East deletion thalassemia

Objective: To analyze the genotype-phenotype correlations among those thalassemia samples with the presence of -α(3.7,) --(SEA) and normal α(2) alleles on their α-globin gene clusters. Methods: Fourteen patients(including 1fetus, 4 males and 9 females, aged 0- 56 years old)who were suspected diagnosed by hematologic analysis and genetic testing among 16 080 participants in our laboratory since from August 2011 to August 2016, were enrolled. Complete blood cell count was performed on XE4000i automatic hemocyte analyzer. HbA0, HbF and HbA2 were tested by high performance liquid chromatography (HPLC). Gap-PCR was adopted to detect three common deletional thalassemia deletions. Reverse dot-blot (RDB) assay was applied for detecting 17 common β-globin gene mutations and three common non-deletional α(2) gene mutations. Two-round nested PCR assay was established to detect the genotype of HKαα in α-thalassemia. Results: Fourteen cases were identified as HKαα/--(SEA) (14/16 080), including a pedigree and a rare case of HKαα/--(SEA) co-inheritance with IVS-Ⅱ-654(C→T) heterozygote. In HKαα/--(SEA) thalassemia group, mean cell volume(MCV) was (69.54±5.92)fl, and mean cell hemoglobin(MCH) was(22.11±2.22)pg and hemoglobin(Hb) was (117.64±18.14) g/L. Compared with normal group, MCV, MCH and Hb in HKαα/--(SEA) thalassemia group, was significantly decreased(P<0.05). There were no significant differences between α-thalassemia control group(--(SEA) /αα) in most hematological parameters (P>0.05). Conclusion: The two-round nested PCR could effectively detect the HKαα/--(SEA) genotype. The hematologic characteristics changed significantly in HKαα/--(SEA) group compared with HbH thalassemia and normal group. The genotype and phenotype non-correlation in patients with α-thalassemia should especially be causious to avoid a misdiagnosis of genetic tests, especially in prenatal diagnosis.

Analysis of Common β-Thalassemia Mutations in North Vietnam

Available and flexible choice of methods for screening and detecting β-thalassemia (β-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common β-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (–TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population.

BackgroundCongenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required.MethodsWe developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates.ResultsThe test was validated through direct sequencing in a blinded study with 100% concordance.ConclusionsThe results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.

Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

Development and evaluation of a reverse dot blot assay for the simultaneous detection of common toxic microalgae along the Chinese coast

Toxic microalgae currently pose a great threat to human health, ecosystem, fishery, tourism, and aquaculture along the Chinese coast. The detection of toxic microalgae by routinely monitoring natural waters is necessary to provide timely mitigation. Therefore, an effective, simultaneous detection protocol should be established for the simple, rapid, and accurate identification of causative algae. This study developed and evaluated a reverse dot blot assay (RDBA) combined with a low-density membrane-based DNA array for the rapid and simultaneous detection of toxic microalgae that are commonly distributed along the Chinese coast. The large subunit rDNA D1–D2 regions of the target species were first sequenced to design taxonomic probes. Probe specificity was validated by performing a cross-reactivity test with dot blot hybridization. The tailed probes were immobilized onto a nylon membrane to prepare a low-density DNA array for RDBA. The established detection procedure involved DNA extraction, biotin (Bio)-labeling of objective sequences by multiple polymerase chain reaction (M-PCR), RDBA, coloration, and judgment of hybridization by the naked eye. Bio-labeled primer-based labeling proved to be an economical and effective method to prepare Bio-labeled PCR products for RDBA. The detection limits of RDBA using the M-PCR-labeling products from DNA templates prepared by different methods were also compared, and a kit-based DNA extraction method displayed the lowest detection limit of 0.5 cells. Simulation results showed that RDBA can recover all target species and was not affected by background DNA. RDBA was proven effective, specific, and sensitive for the simultaneous detection of toxic microalgae in the field samples. Therefore, this method may be used in the field monitoring of natural samples.

Identification of Nondeletional α-Thalassemia in a Prenatal Screening Program by Reverse Dot-Blot in Southern China

The aim of this study was to demonstrate the performance of nondeletional α-thalassemia (α-thal) prevention using a reverse dot-blot method at a Mainland Chinese hospital. A prenatal control program for nondeletional Hb H disease was performed between January 2009 and December 2013. All couples were screened for α-thal trait, and for couples in which one partner tested positive for α0-thal, the other was subjected to screening for Hb Constant Spring (Hb CS, HBA2: c.427T > C) and Hb Quong Sze (Hb QS, HBA2: c.377T > C) mutations by reverse dot-blot assay. Prenatal diagnoses were offered in at-risk pregnancies. During the study period, 51,105 couples were found to be carrying α-thal; among these, 35 (0.07%) couples were found to be at-risk of conceiving an offspring with nondeletional Hb H disease, including 25 couples for Hb H-CS and 10 cases for Hb H-QS. Nine fetuses were diagnosed with nondeletional Hb H disease, and eight of the affected pregnancies were terminated. Detection of nondeletional α-thal is necessary for any prenatal diagnosis (PND) programs in Southeast Asian countries. Reverse dot-blot is a relatively simple method for simultaneous typing of common nondeletional α-thal mutations.

Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot

To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). Optimised conditions were as follows: tailed hybridization probes (1–2pmol/l) were immobilized on a membrane by baking for 2h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1μg/l of 2% RRS and 0.5ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively.

Prenatal diagnosis of β-thalassemia in Guangxi Zhuang Autonomous Region, China

The aim of this study was to establish a comprehensive prenatal diagnosis service and to control the birth of thalassemia children in Guangxi Zhuang Automonous Region, China. Prenatal diagnosis was performed in 1,058 couples with 'at risk' β-thalassemia from Guangxi Zhuang Automonous Region. Fetal samplings were collected by chorionic villus sampling in the first trimester, by amniocentesis in the second trimester and by cordocentesis in the third trimester. DNA analysis was carried out using polymerase chain reaction, reverse dot blot assay, multiplex ligation-dependent probe amplification method and DNA sequencing. Automated high-performance liquid chromatography system was used to analyze the fetal hemoglobin in pregnancies in case mutations were unidentified. A total of 12 different β-thalassemia mutations were characterized from 2,116 parents. The most common mutation for β-thalassemia was CD41-42 (-CTTT) followed by CD17 (A→T). Prenatal testing revealed 315 normal fetuses, 500 carriers and 253 β-thalassemia major fetuses. The couples having fetuses with β-thalassemia major were counselled to terminate the pregnancies. Postnatal follow-up confirmed all pregnancies. Our prenatal diagnosis strategy proved to be highly effective in reducing severe thalassemia in pregnant populations.

A reverse dot blot assay for the expanded screening of eleven Chinese G6PD mutations

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is a multiethnic inherited disease with a particularly high prevalence in tropical and subtropical regions including southern China. A convenient and reliable method is required to detect common G6PD mutations in the Chinese population. MethodsWe developed a reverse dot blot (RDB) assay for the expanded screening of eleven mutations (c.95A>G, c.392G>T, c.871G>A, c.1004C>T, c.1004C>A, c.1024C>T, c.1360C>T, c.1376G>T, c.1381G>A, c.1387C>T, c.1388G>A). The method consists of a single-tube multiplex PCR amplification of four fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a group of 213 unrelated Chinese patients. ResultsThe test had a detection rate of 95.8%, validated by direct sequencing in a blind study with 100% concordance. ConclusionsThe results demonstrate that our reverse dot blot assay is an easy, reliable, high-yield and cost-effective method for genetic screening to identify G6PD patients and carriers among the Chinese population.

Comparison between probe melting curve analysis based on real-time fluorescent PCR and reverse dot blot assay for gene and prenatal diagnosis of β-thalassemia

Objective To evaluate the reliability of the probe melting curve analysis (PMCA) based on real-time fluorescent PCR assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia. Methods A total of 135 cases of peripheral blood samples were collected from Zhuhai Municipal Maternity and Child Healthcare Hospital between 2008 and 2010.All the samples were performed preliminary diagnosis according to the hematological data.Of these, 119 cases were diagnosed as β-thalassemia trait, 4 cases were diagnosed as severe thalassemia and 12 cases were normal.In addition, 44 cases of amniotic fluid and 8 cases of cord blood with high-risk for severe β-thalassemia were also collected.The diagnostic reliability of the PMCA assay and reverse dot blot assay was evaluated on 187 above-mentioned cases by direct DNA sequencing analysis in a double-blind study. Results The genotypes of 185 cases were detected accurately based on the PMCA assay except for two cases:one heterozygote with Ini(ATG>AGG) was omitted and another heterozygote couldn′t be distinguished between CD43(G>T) and CD37(G>A).For the RDB assay, only one heterozygote with CD71-72(+T) was not detected accurately in the above-mentioned cases.Compared with the DNA sequencing analysis, the sensitivity, specificity, negative predictive value, positive predictive value and diagnostic efficiency of the PMCA assay were 98.75%, 100.00%, 93.10%, 100.00% and 98.93%, respectively.The corresponding value of the RDB assay were 99.38%, 100.00%, 96.42%, 100.00% and 99.47%, respectively.There were no significant between-group differences in the diagnostic efficiency of the two assays (P>0.05).The results of prenatal diagnosis were in complete concordance with the follow up results, after the birth or induced labour of the fetuses. Conclusions The PMCA assay can be used as an alternative and verified method of RDB assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia.(Chin J Lab Med, 2012，35：413-417) Key words: beta-Thalassemia; Polymerase chain reaction; Melting curve analysis; Nucleic acid hybridization; Prenatal diagnosis