Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System.

BACKGROUND: Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. METHODS: In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. RESULTS: The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. CONCLUSION: Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens.

Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification.

Rotaviruses, noroviruses and astroviruses are the major viral pathogens leading to diarrhea worldwide. Epidemiological investigations of outbreaks associated with these viruses have been impeded by the lack of methods for quick diagnosis and detection. In the current study, a multiplex real-time nucleic acid sequence-based amplification (RT-NASBA) system was developed for the simultaneous detection of rotavirus A/norovirus genogroup II/astrovirus. The specificity and sensitivity of the assay were compared with multiplex RT-PCR. The results showed that the multiplex RT-NASBA assay was established successfully, and robust signals could be observed in 10 minutes with high specificity. The limit of detection of the multiplex RT-NASBA assay was 7, 100, and 200 copies per reaction for rotavirus A, norovirus genogroup II, and astrovirus, respectively. The assay was thus 10 to 100 times more sensitive than multiplex RT-PCR. Clinical evaluation indicated that the assay was 100% concordant with multiplex RT-PCR and was reliable for the detection of both single infections and multiple infections in stool samples. To the best of our knowledge, this is the first multiplex RT-NASBA assay established for the detection of three major diarrhea-causing viruses. This assay provides a valuable platform for the rapid, specific, sensitive and simultaneous diagnosis of these pathogens, especially in resource-limited countries where expensive thermocycling equipment is not available.

The establishment and clinical evaluation of a novel, rapid, no-wash one-step immunoassay for the detection of dengue virus non-structural protein 1.

Dengue fever is a highly endemic arthropod-borne viral disease in the tropical and sub-tropical countries and is rapidly becoming a global threaten. Diagnosis has been conducted by either traditional serological methods or molecular biological techniques. However, these methods are either labor-intensive, time-consuming or with multiple steps, which are not suitable for high throughput detection of large quantity of samples. In the current study, a novel, rapid, no-wash one-step amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) was developed and optimized for the diagnosis of dengue fever through the detection of dengue virus non-structural protein 1 (NS1). The linear range of the assay was determined to be 60,000 pg/mL to 200 pg/mL, with a lower detection limit of 127.45 pg/mL for NS1 protein. The precision of the assay was 8.24 % and 4.93 % for the high and low concentration. Clinical evaluation indicated that the sensitivity and specificity of the assay was 91.49 % and 81.54 %, respectively. This novel, rapid, no-wash one-step AlphaLISA assay is convenient and sensitive, which could be a good alternative for the screening of dengue fever in a high throughput format.

[A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province].

OBJECTIVE: To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Complete nucleotide sequence analysis of the norovirus GII.17: A newly emerging and dominant variant in China, 2015.

Norovirus is an important pathogen which accounts for majority of the viral related acute gastroenteritis. Recently, a variant of genotype GII.17 was reported to be predominant over GII.4 and accounted for several acute gastroenteritis outbreaks in Asia. In the current study, the full genome of a norovirus strain ZHITHC-12 isolated during this outbreak period in China was identified and characterized. The viral genome was 7557 nucleotides in length and a phylogenetic analysis based on full length genome sequences indicated that ZHITHC-12 belonged to GII.17 genotype. A further phylogenetic analysis based on all available polymerase and capsid sequences showed that ZHITHC-12 was in Cluster III on both phylogenetic trees and grouped with other strains also isolated during 2013 to 2015. Moreover, homology modeling analysis based on GII norovirus capsid 5BSX template revealed that substitutions, mutations, and more importantly, deletions and insertions, occurred at or near the putative epitopes and histo-blood group antigen (HBGA) binding sites in its protruding P2 domain, which might confer new antigenic or biological properties for this novel variant. In summary, the first full genome and capsid protein structure of a novel norovirus GII.17 variant isolated in China was extensively characterized. The data would be helpful not only for the epidemiology study, but also for the diagnostic tool development and effective vaccine design in the future.

HIV vaccine research: the challenge and the way forward.

Human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is a worldwide epidemic, with over 35 million people infected currently. Therefore, the development of a safe and effective HIV-1 vaccine is on top of the global health priority. In the past few years, there have been many promising advances in the prevention of HIV/AIDS, among which the RV144 Thai trial has been encouraging and suggests optimization of the current vaccine strategies or search for novel strategies. Here we reviewed the brief history of HIV-1 vaccine, analyzed key challenges existing now, and illustrated future research priority/directions for a therapeutic or prophylactic HIV-1 vaccine, with the hope of accelerating the speed of vaccine development. We believe that an effective HIV-1 vaccine, together with other prevention approaches, will bring an end to this epidemic in the near future.

Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

Reliable and high-throughput mutation screening for beta-thalassemia by a single-base extension/fluorescence polarization assay.

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.

[Rapid diagnosis of non-deletion alpha-thalassemias by reverse dot blot].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Accurate and rapid prenatal diagnosis of beta-thalassemia by a multiplex primer extension and denaturing high-performance liquid chromatography technique].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: The PE-DHPLC protocol can be a simple, rapid, and highly accurate assay in the prenatal detection of common beta-thalassemia mutations.

[A rare transcription mutation (-90 C-->T) in a Chinese family with beta-thalassemia].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Molecular epidemiological study of alpha- and beta-thalassemia in Sihui city.

OBJECTIVE: To investigate alpha- and beta-thalassemia (alpha- and beta-thal) gene frequencies and gene mutation spectrum in the population of Sihui City. METHODS: The umbilical cord blood samples from 1 007 neonates and peripheral blood samples from 1 524 apparently healthy adults for pre-marriage health check in Sihui city were collected for molecular epidemiologic study of alpha- and beta-thal respectively. The diagnostic standard for alpha-thal was the presence of Hb Bart's, and that for beta-thal was both the decrease of mean corpuscular volume (MCV<80 fl) and the increase of Hb A(2) level (> or = 3.5%). The samples of identified subjects with positive thal genotypes were further examined with PCR-based DNA analysis for determining the alpha- or beta-globin gene genotype, while those from subjects with positive genotypes but without mutations known to Chinese subjects were subjected to DNA sequence analysis of beta-globin gene. In addition, the alpha-thal alleles, -alpha(3.7) and -alpha(4.2) were examined in all umbilical cord blood samples. RESULTS AND CONCLUSION: Of all the 1 007 umbilical cord blood samples, 110 were identified as from alpha-thal gene carriers, 3 from patients Hb H disease and 1 from patients with hydrops fetalis, which meant an alpha-thal gene frequency of 11.72% (118/1 007). Three types of alpha-gene deletion were identified in this cohort, with the frequency of 53.4% (--SEA)), 34.7% (-alpha(3.7)) and 11.9% (-alpha(4.2)) respectively. By examining the peripheral venous blood samples from the 1,524 healthy adult subjects, 59 subjects were found to be beta-thal gene carriers with a rate of 3.87% (59/1,524), whose genotypes were determined and from whom 7 beta-thal mutations were identified. Of these 59 beta-thal gene carriers, 11 were diagnosed as having heterozygotes compound for beta- and alpha-thal genes with the deletion of the --(SEA) in 7 cases and -alpha(3.7) in 4 cases respectively, showing an incidence of 0.72% (11/1,524). The three commonest point mutations, beta CD41-42 (-CTTT) frameshift mutation, beta IVS2-654(C-->T) aberrant splicing mutation and beta-28 (A-->G) transcription mutation occurred with a total frequency of 84.75% among subjects with beta-thal allele mutations. In addition, a novel mutation, beta-globin gene promoter -90 (C-->T) allele was detected for the first time in Chinese subjects.

Evaluation of clinical application of gap-PCR as a routine method for alpha-thalassemia carrier detection.

OBJECTIVE: To evaluate the feasibility of using gap-PCR for routine screening of alpha-thalassemia in clinical laboratory. METHODS: A total of 382 clinical blood samples randomly collected from the population of Zhuhai city were screened for alpha-thalassemia determinants with hematological and gap-PCR method respectively in a double-blind manner. Parallel analysis with Southern blotting was performed to verify the genotyping results by PCR. RESULTS: Of the 382 samples tested, 3 common alpha-thalassemia genes with genotypes of --(SEA)/alpha alpha, -alpha(3.7)/alpha alpha and -alpha(4.2)/alpha alpha were detected in 21 (5.50%), 7 (1.83%) and 3 (0.79%) cases respectively by gap-PCR, including 7 cases with normal phenotype and 3 case of iron-deficiency anemia. The overall incidence of alpha-thalassemia was 8.12% in the population of Zhuhai city, as determined by gap-PCR, in total agreement with the results by Southern blotting. Only 21 of the 31 alpha-thalassemia cases were identified by hematological analysis (besides 2 cases with alpha-thalassemia phenotype undetermined), which had a false-negative rate of 32.3%. Seven silent alpha-thalassemia and 3 mild alpha-thalassemia cases failed to be detected by hematological analysis, resulting in a rate of 2.62% for failure of detection. CONCLUSION: Gap-PCR method is specific and feasible as a better alternative for alpha-thalassemia screening, especially advantageous in detecting silent carriers in comparison with hematological method.

[Rapid detection of novel avian influenza virus subtype H7N9 by multiplex real-time RT-PCR].

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].

OBJECTIVE: To explore the epidemiological characteristics of viral diarrhea of norovirus (NV), sapovirus (SV) and astrovirus (AstV) among children in Zhuhai during winter and spring. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α-thalassemia in maternal plasma.

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.

[Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning].

OBJECTIVE: To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[One-step multiplex RT-PCR for rapid screening of type A, B and novel A (H1N1) influenza viruses].

OBJECTIVE: To developed a multiplex RT-PCR assay for simultaneous screening of type A, B and novel A (H1N1) influenza viruses. METHODS: 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. RESULTS: 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%. CONCLUSION: 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.

[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].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.