Rapid detection of novel avian influenza virus subtype H7N9 by multiplex real-time RT-PCR / 病毒学报

In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.

Epidemiological features on 3 important viral diarrhea among children in Zhuhai during winter and spring / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the epidemiological characteristics of viral diarrhea of norovirus (NV), sapovirus (SV) and astrovirus (AstV) among children in Zhuhai during winter and spring.</p><p><b>METHODS</b>Stool specimens were collected from children with viral diarrhea in Maternal and Child Health Hospital of Zhuhai from November 21, 2009 to April 3, 2010. Nucleic acid of NV, SV and AstV from negative specimens of rotavirus and adenovirus were detected by using Reverse transcription-polymerase chain reaction (RT-PCR), and the types of positive samples of NV were also classified at the same time.</p><p><b>RESULTS</b>The total detection rate of the three viruses is 21.49 percent, the highest detection rate is 29.05% in December 2009, the lowest detection rate is 12.20% in February 2010, 87.96% of positive specimens were from children patients aged from 0 to 30 months. The season detection rate of NV, SV and AstV are 14.70%, 2.75% and 4.04% respectively. There were significant differences of NV and SV detection rates in every month of the season, whereas the AstV detection rate was comparatively stable. The highest detection rate of NV is 34.09% in children patients aged from 12 to 18 months, the highest SV detection rate is 12.5% in children patients aged from 60 to 120 months, and the highest AstV detection rate is 16.67% in children patients aged from 24 to 30 months. All the NV were belong to G II genogroup.</p><p><b>CONCLUSIONS</b>NV is one of the main pathogens causing viral diarrhea among children in Zhuhai during winter and spring, SV and AstV are also important pathogens. So we should strengthen the monitoring of viral diarrhea caused by NV, SV and AstV in infants and young children.</p>

Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria.</p><p><b>METHODS</b>Type-specific PCR primers labeled with biotin and oligonucleotide probes were designed according to the conservative genes of 9 major foodborne bacteria Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7 (Stx1 and Stx2), Shigella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus. A one-tube multiplex PCR system for simultaneous amplification of these bacteria was established, and the DNA probes were spotted and immobilized in the wells of the plate in 5x5 array format. Stable hybridization system between PCR products and oligonucleotide probes in the microwell was established after condition optimization. Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products.</p><p><b>RESULTS</b>Twenty standard bacteria strains were used to validate the 96 microwell plate DNA diagnostic chip and highly specific and stable experiment results were obtained. Using this chip assay, the causal pathogen Staphylococcus aureus was identified within 12 h after the sampling from an incident of food poisoning, and the result was consistent with that obtained using conventional bacterial culture and biochemical identification.</p><p><b>CONCLUSION</b>The novel 96 microwell plate DNA diagnostic chip allows rapid, accurate, automated and high-throughput bacterial detection and is especially valuable for quick response to such public health emergencies as food poisoning.</p>

One-step multiplex RT-PCR for rapid screening of type A, B and novel A (H1N1) influenza viruses / 南方医科大学学报

<p><b>OBJECTIVE</b>To developed a multiplex RT-PCR assay for simultaneous screening of type A, B and novel A (H1N1) influenza viruses.</p><p><b>METHODS</b>Two pairs of universal primers in were designed for amplifying the M gene and NS gene of type A and B influenza viruses, respectively. A pair of specific primers of HA gene was designed to detect novel A (H1N1) influenza virus. A one-step method was used to establish the multiplex RT-PCR system. A blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. The clinical practicability and efficacy of this assay was also evaluated.</p><p><b>RESULTS</b>The RT-PCR products were analyzed using agarose gel electrophoresis, which yielded distinct bands of the target fragments without non-specific reactions, suggesting the high efficiency and specificity of the multiplex RT-PCR. Blinded study of 50 samples demonstrated a concordance rate of 100%.</p><p><b>CONCLUSION</b>This multiplex RT-PCR assay allows one-step simultaneous detection of type A, B and novel A (H1N1) influenza viruses rapidly and accurately, and provides a valuable low-cost screening technique for influenza epidemic monitoring and early diagnosis.</p>

A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province.</p><p><b>METHODS</b>Couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled in this prospective screening program, which was supported by the two-level network composed of 6 local hospitals for testing thalassemias and follow-up for genetic counseling. A conventional heterozygote screening strategy was used to determine alpha and beta thalassemia traits in women and their partners according to the standard procedures of hematological phenotype analysis. Then confirmative diagnosis of alpha and beta thalassemia was performed on those couples suspected at-risk for severe thalassemia by using the PCR-based molecular diagnostic assays. The couples at-risk for severe thalassemia were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus.</p><p><b>RESULTS</b>During the period between January 1998 and December 2005, the screened records included 85522 young females and their partners for premarital screening and 10439 pregnant women for prenatal screening, with 71.38% coverage of total population recorded in this city for premarital screening. Six thousands five hundreds and sixty-three individuals in total were found to be the carriers of thalassemias, with 4312 for alpha thalassemia (4.5%) and 2251 for beta thalassemia (2.3%), respectively. One hundred and forty-eight couples were diagnosed to be at-risk for thalassemias, including 103 for alpha thalassemia and 45 for beta thalassemia, respectively. Successful prenatal diagnosis was made for 142 (98 for alpha thalassemia and 44 for beta thalassemia) out of 148 (95.9%) pregnancies at-risk for severe thalassemias. Twenty-three cases of hydrops fetalis, 4 of Hb H diseases and 14 of beta thalassemia were identified. All 41 pregnancies with affected fetuses were voluntarily terminated. Thus, this has led to a marked decrease of severe thalassemia syndrome since the program started.</p><p><b>CONCLUSION</b>We presented the first community-based prospective screening program in China for control of alpha and beta thalassemia in Zhuhai city with a population of 1.29 million through premarital or prenatal screening. This model could be used for control of thalassemias and other hemoglobinopathies in other regions of China and also in other developing countries.</p>

A rare thalassemia intermedia case caused by co-existence of Hb H disease (--(SEA)/-alpha(4.2)) and beta-thalassemia major (beta (CD17A)>T/beta (IVS2-654C)>T): implications for prenatal diagnosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the relation between the genotype and phenotype in a Chinese patient with thalassemia intermedia and its implications for prenatal diagnosis and genetic counseling of thalassemia intermedia caused by co-existence of Hb H disease and beta; thalassemia major.</p><p><b>METHODS</b>Phenotypic analysis was performed using standard hematological tests to measure red blood cell parameters and Hb concentration. Genotyping of beta thalassemia mutations and alpha thalassemia deletion were conducted using reverse dot-blot (RDB) assay and gap-PCR, respectively. We investigated the pathogenesis of this case by genotype-phenotype correlation analysis based on screening of the patient's family members. Prenatal diagnosis for a high-risk fetus in this family was performed by amniotic fluid DNA analysis.</p><p><b>RESULTS</b>The proband was identified as a patient with severe thalassemia intermedia caused by co-existence of Hb H disease (--(SEA)/-alpha (4.2)) and beta-thalassemia major (beta (CD17A)>T/beta (IVS2-654C)>T), whose father was heterozygous for beta thalassemia (beta (CD17A)>T/beta (N)) and alpha-thalassemia trait (--(SEA)/) and the heterozygous for beta thalassemia (beta (IVS2-654C)>T / beta (N)) and silent alpha-thalassemia (-alpha (4.2)/). The result of prenatal diagnosis showed co-existence of beta thalassemia major and silent alpha thalassemia in the high-risk fetus, and the parents requested termination of pregnancy after genetic counseling.</p><p><b>CONCLUSION</b>We report for the first time a rare thalassemia intermedia case resulting from 4 complex alpha/beta thalassemia combination and the molecular pathogenesis of thalassemia intermedia is updated in the Chinese population. The practice of prenatal diagnosis in this case may also provide reference for diagnosis of similar cases.</p>

Accurate and rapid prenatal diagnosis of beta-thalassemia by a multiplex primer extension and denaturing high-performance liquid chromatography technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a primer-extension in combination with denaturing high-performance liquid chromatography (PE-DHPLC)-based assay for prenatal diagnosis of the five most common beta-thalassemia mutations in Chinese.</p><p><b>METHODS</b>The human beta-globin gene fragment was amplified by PCR, followed by a multiple PE reaction specific for each five mutations. Then the PE product mixtures were separated for genotyping of beta-globin gene mutations using fully-denaturing DHPLC analysis.</p><p><b>RESULTS</b>In a blind study, prenatal diagnosis was performed on thirty-six at-risk families for beta-thalassemia major. Reverse dot blot (RDB) analysis was used to validate each result, showing an accuracy rate of 100% for PE-DHPLC in a total of 108 samples tested. Overall, by PE-DHPLC analysis, the authors could identify the genotypes involving the five mutations and normal alleles corresponding to 94.4% (102/108) and actually make final decision for prenatal diagnosis covering 97.2% (35/36).</p><p><b>CONCLUSION</b>The PE-DHPLC protocol can be a simple, rapid, and highly accurate assay in the prenatal detection of common beta-thalassemia mutations.</p>

Rapid diagnosis of non-deletion alpha-thalassemias by reverse dot blot / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a rapid and convenient method of reverse dot blot (RDB) analysis for detecting the point mutations of non-deletion alpha-thalassemia in Chinese.</p><p><b>METHODS</b>Label biotin to primers and amplify human alpha2 globin gene selectively, then hybridize PCR products with a set of oligonucleotide probes immobilized on strips, and develop colour to detect non-deletion alpha-thalassemia defects.</p><p><b>RESULTS</b>The PCR system using biotin-labeled primers could specifically amplify a 1085 bp fragment of human alpha2 globin gene which encompasses all six alpha-thalassemia mutations. After being hybridized with sequence-specific oligonucleotide probes and colour development, it could simultaneously identify all six types of non-deletion alpha-thalassemias encountered in Chinese.</p><p><b>CONCLUSION</b>This method does not need semi-nested PCR, and the products amplified by biotinylated primers can be used directly to hybridize with the probes on strips under the identical conditions of hybridization. So, it is a specific and multiplex detection assay for screening non-deletion alpha-thalassemia defects in Chinese.</p>

A rare transcription mutation (-90 C-->T) in a Chinese family with beta-thalassemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a rare transcription mutation (C-->T) at position -90 of the beta-globin gene previously unreported in the beta-thalassemia carriers from a Chinese family.</p><p><b>METHODS</b>In phenotype analysis, standard hematological techniques were used to measure RBC counts and Hb concentration. Reverse dot blot (RDB) analysis, which can simultaneously detect 18 known types of beta-thalassemia mutations in Chinese, was used to scan beta-globin gene mutations. DNA sequence analysis of the entire human beta-globin gene was performed to characterize the underlying causative mutation of the sample and to identify its genotype. A semi-quantitative RT-PCR method was used to measure beta-globin gene expression in the form of mRNA from the subjects.</p><p><b>RESULTS</b>The proband, his brother and his mother presented a typical beta-thalassemic trait with reduced mean corpuscular volume (MCV, 68.2-73.6 fL) and elevated level of Hb A(2) (5.7%-6.4%) but no known beta-thalassemia mutations were found in the samples by RDB analysis. DNA sequencing of the beta-gene region of these three samples revealed heterozygosity for the C-->T substitution at position -90 within proximal CACCC box of the beta-globin gene promoter element, which was previously unreported in the Chinese population. Analysis of mRNA from the positive carriers demonstrated that the mutant beta-globin gene significantly reduced beta-globin transcription (mutants: 2.233 +/- 0.01 vs normal: 3.779+/-1.19; 95%CI: 3.060, 4.499), showing a level comparable with that of the other beta-thalassemia heterozygotes (2.110+/-0.53, 95%CI: 1.732, 2.488).</p><p><b>CONCLUSION</b>A rare transcriptional mutation that led to beta-thalassemia in Chinese population has been characterized. The findings enrich knowledge of the mutation spectrum of beta-thalassemia.</p>