HAND system-based four multiplex RT-PCR for simultaneous detection of four diarrhea viruses / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.</p><p><b>METHODS</b>Primers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.</p><p><b>RESULTS</b>The 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.</p><p><b>CONCLUSION</b>The established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.</p>

Rapid detection of three common deletional alpha thalassemias in Chinese by single-tube multiplex PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population.</p><p><b>METHODS</b>Four groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation.</p><p><b>RESULTS</b>Homozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families.</p><p><b>CONCLUSION</b>The single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics.</p>

Prevalence survey and molecular characterization of alpha and beta thalassemia in Liuzhou city of Guangxi / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the gene frequencies and mutation patterns of alpha thalassemia (alpha-thal) and beta thalassemia (beta-thal) in Liuzhou city of Guangxi Zhuang Autonomous Region.</p><p><b>METHODS</b>Cluster sampling was used. A total of 1 028 of umbilical blood samples were collected for a prevalence study of alpha-thal and a total of 1 312 healthy young people when receiving pre-marriage consultation were recruited for a beta-thal prevalence survey. Individuals live in city or town area of Liuzhou. A complete blood count as well as hemoglobin electrophoresis analysis were done in all of samples for phenotyping of alpha and beta-thals. Those with Hb Bart's for alpha-thal indicator and those with both microcytosis (MCV < 85 fl) and elevated levels of Hb A(2) (>/=4.0%) for beta-thal were further studied by DNA analysis. PCR-based methodologies were used to characterize the mutation contributions of alpha and beta-thals. All the subjects were tested for the state of carrying beta-thala alleles for evaluating the situation of the compound heterozygotes of alpha-thal with beta-thal.</p><p><b>RESULTS</b>Of 1 028 random samples of umbilical blood screened, 112 of subjects were defined to be the gene carriers of alpha-thal. The alpha-thal carrier rate was as high as 11.19% including 3 compound heterozygotes. Five well-known types of alpha-thal alleles were detected with gene contributions of 37.4% (--(SEA) deletion), 31.3% (-alpha(3.7) deletion), 17.4% (-alpha(4.2) deletion), 12.1% (alpha(CS)alpha mutation), and 0.9% (alpha(QS)alpha mutation), successively. Of the 1 312 adult specimens studied, 89 with beta-thal including 14 of the compound higher Hb F subjects were detected. All of the 89 phenotypic beta-thal carriers had the mutations in the beta-globin gene, making the overall prevalence 6.78%. The commonly seen three mutations, beta CD41 - 42 (-CTTT) frameshift, beta CD17 (T-A) nonsense mutation and beta-28 (A-G) promoter variation were accounted for 90% of the beta-thal alleles in Liuzhou. Of these beta-thal subjects, 16 (accounting for 18%) were found to be the compound heterozygosity for a beta-thal and an alpha-thal with 9 different types of gene defects with a detection rate 1.22%.</p><p><b>CONCLUSION</b>Data from ecidation of alpha and beta-thal gene frequencies and mutation spectrum in Liuzhou city was useful for genetic counselling and prenatal diagnosis of this disease.</p>

An improved PCR-based megaprimer method for site-directed mutagenesis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM).</p><p><b>METHODS</b>This protocol is based on the design of two different plasmid DNA templates. Template 1 lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. This modification avoids amplification of full-length wild-type sequences while two templates, the forward and reverse flanking primers exist simultaneously in one reaction tube. A megaprimer is synthesized in the first PCR reaction using template 1, forward primer and mutagenic primer. The megaprimer that needn't the cumbersome gel purification step is directly added to the second PCR reaction system. The second PCR reaction(PCR 2) containing two stages is performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer is extended to form the full-length mutation product. In the second stage of PCR 2, the extended megaprimer containing SDM residues is subsequently amplified with the two flanking primers. All of the final PCR products contained the desired mutation.</p><p><b>RESULTS</b>Fifteen types of rare beta-thalassemia mutations in Chinese were obtained using this method. Each of these modified fragments was separately cloned into the pGEM-T vector and sequenced. The desired mutations involved in mutagenesis amplicons were identified in all clones.</p><p><b>CONCLUSION</b>This improved PCR-based megaprimer method for site-directed mutagenesis is rapid, simple and highly efficient, and the success rate of mutagenesis could reach 100%. Furthermore, this method is suitable for routine application in molecular cloning.</p>