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

Background: and 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 and on a field-deployable PCR system was developed.

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.

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.

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.

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.

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.

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.

[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.

[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.

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.

[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.

[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 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.

[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.

A novel mutation of -73(A-->T) in the CCAAT box of the beta-globin gene identified in a patient with the mild beta-thalassemia intermedia.

The beta-thalassemia is one of the most common autosomal recessive disorders in Southern China. The point mutation of beta-globin gene is the commonest molecular pathogenic mechanism. In Chinese population, over 30 mutations have now been identified.

Rapid, accurate genotyping of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 based on the use of denaturing HPLC.

The genotypes of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are related to alcohol dependence and some human disorders. Rapid, accurate genotyping methodologies for specific polymorphisms of these two genes are needed for molecular screening and testing of alcohol-related problems in populations. A polymerase chain reaction (PCR) composed of two separate amplicons was designed to generate sequences containing the polymorphic site of interest in the ADH1B and ALDH2 genes.

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.

Carrier screening for alpha- and beta-thalassemia in pregnancy: the results of an 11-year prospective program in Guangzhou Maternal and Neonatal hospital.

Objectives: To evaluate the first prospective screening program in China for control of alpha and beta-thalassemia in the population of pregnant couples.

Methods: During the period between January 1993 and December 2003, a hospital-based preventive program was conducted at the biggest birth center in Guangzhou, with 1/17 of all deliveries in this city referred annually by use of conventional heterozygote screening strategy in combination with the system of regular healthcare examination in pregnancy.

Results: The screened records included 49 221 pregnant women, and 4503 husbands of the pregnant women showed positive on the screening test.

[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.

[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.

[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.

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.

Rapid, accurate genotyping of beta-thalassaemia mutations using a novel multiplex primer extension/denaturing high-performance liquid chromatography assay.

Beta-thalassaemia is a common inherited disorder of haemoglobin synthesis worldwide, with an estimated 3-10% frequency in certain regions. Rapid, accurate genotyping methodologies for specific, causative mutations of the beta-globin gene are needed for pre- and postnatal screening and diagnosis of this disease in different ethnic populations. In this study, we performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) for simultaneously detecting and genotyping the five most common molecular lesions in the beta-globin gene [codons (CDs) 41-42 (-TCTT), IVS-2-654 (C-->T), - 28 (A-->G), CD17 (A-->T) and CD71-72 (+ A)] in Chinese populations.

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.