OBJECTIVETo establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.

METHODSAccording to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients.

RESULTSThe HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients.

CONCLUSIONThe TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.

Hepatitis A virus ; genetics ; isolation & purification ; Humans ; Real-Time Polymerase Chain Reaction ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVEReal-time RT-PCR, cell culture and embryonated eggs culture for influenza detection were compared by analyzing the data of influenza surveillance in Shenzhen in second half of 2009.

METHODS1092 clinical samples (throat swabs) collected during second half of 2009 were tested by real-time RT-PCR, cell culture and embryonated eggs culture, and the results were analyzed by statistical methods.

RESULTSThe positive rate were 54.21%, 27.11% and 16.21% using real-time RT-PCR, cell culture and embryonated eggs culture, and the sensitive were 100%, 50% and 29.9%. The lowest dilutions of virus detected by real-time RT-PCR were 10(-2) TCID50/ml.

CONCLUSIONThe sensitive of real-time RT-PCR was higher than culture and the specificity was also very high. It was more suitable for emergency detect. The sensitive of cell culture for H3N2 subtype was higher, and sensitive of embryonated eggs culture for type B was higher.

Animals ; Chick Embryo ; Humans ; Orthomyxoviridae ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Virus Cultivation ; methods

OBJECTIVETraditional prenatal diagnosis for congenital diseases were villus sampling and amniocentesis. These invasive diagnosis methods are not only technical complicated, but also harmful to mother or fetus. Fetus in its different gestational age has its different type of hemoglobin or different amount of hemoglobin, especially epsilon hemoglobin exiting in the body of 10 weeks gestation fetal, however gamma hemoglobin has its high amount before baby to be born. But epsilon and gamma hemoglobin did not exist in the bodies of adults bodies. It is possible to use advanced molecular biological technique to extract the fetal hemoglobin gene from maternal peripheral blood. In articles from domestic and abroad, no report related to fetal hemoglobin extraction from maternal peripheral blood was found. We tried to use non-invasive method to detect fetal hemoglobin epsilon/gamma gene from maternal peripheral blood by molecular biological technique. The purpose was to establish a convenient, sensitive and special method to be a basis of screening prenatal diseases in the population and lay a basis for family planning and clinical application.

METHODSBlood samples were collected and the fetal mRNA extracted from the pregnant women with the use of random primer. We used ultraviolet spectrophotometer to test the concentration and purity of extracted mRNA are suitable for reverse transcription. Reverse transcription of mRNA into cDNA was carried out and cDNA by PCR with the special epsilon/gamma primer being used. Via 1.2% EB in agarose gel electrophoresis, we used "Gel Works System" to scan the electrophoresis image to detect epsilon/gamma gene band.

RESULTSThe peripheral blood of pregnant women was collected. With RT-PCR and agarose gel electrophoresis method, we detected epsilon/gamma gene successfully in 7 samples with 6 positive and 1 negative.

CONCLUSIONThis was the first time that we used non-invasive way to detect expression of fetal epsilon/gamma gene in maternal blood to have found that this was a simple method to separate fetal cells from maternal blood, and could easily be accepted by pregnant women. Success of RT-PCR to detect fetal specific mRNA gave the hint that this method could be used in the field of prenatal diagnosis of hemoglobin disease, predicting fetal gender, predicting Rh blood type and single gene disease and be used widespread in prenatal diagnosis.

Female ; Globins ; genetics ; Humans ; Pregnancy ; blood ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; methods

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.
Gastroenteritis ; virology ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Viruses ; isolation & purification

OBJECTIVETo establish the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods for on-site HIV-1 detection.

METHODSAs for the real-time fluorescent RT-LAMP, we firstly tested the specificity and sensitivity, then explored its quantitative determination, and finally applied the method to the detection of 35 HIV-1 positive samples. For colorimetric judgment, after choosing different ameliorates to modify Hydroxynaphthol blue (HNB), we tested their real effects on coloration, and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.

RESULTSThe real-time fluorescent RT-LAMP showed great specificity of HIV-1, and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction. On testing 35 HIV-1-positive samples, the method could reach 100 percent detection rate. However, for the quantitative determination, the quantitative relation was not observed regarding the HIV-1 RNA of below 10(3) copies per reaction. Three modified HNB dyes with clear color variation between the reaction tubes of the negative and the positive were got in the study, and their sensitivities equaled to the level of agarose gel electrophoresis. Similarly, 100% (35/35) detection rate was reached when the colorimetric RT-LAMP with the modified dyes was applied to detect 35 HIV-1-positive samples.

CONCLUSIONThe established real-time fluorescence method and the modified color judgment of RT-LAMP could be helpful for truly achieving rapid, accurate, and sensitive on-site detection of HIV-1.

HIV-1 ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo get an insight into the molecular mechanisms of diseases development and targeted therapy at the transcriptome level and search for potential therapeutic targets.

METHODSThe present researchers established a cDNA microarray platform and applied component plane presentation integrated self-organizing map (CPP-SOM) to the microarray data obtained from a differentiation model, all trans retinoic acid-induced differentiation in NB4 cells.

RESULTSThe platform included 12630 unique clones, including 9436 known genes. By CPP-SOM, the researchers were able to not only well classify the regulated genes into functionally distinct categories but also depict transcriptional changes throughout the process of the development of diseases or drug treatment.

CONCLUSIONThe platform has proven to be steady and reliable, and the CPP-SOM could serve as an important and good tool for analysis of microarray data.

Cell Line, Tumor ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEA new simple RT-LAMP method was applied to detect measles virus nucleic acid and compared with nest-RT-PCR.

METHODSCompare the detection rate of the RT-LAMP method with that of nest-RT-PCR by detecting measles virus nucleic acid from measles virus and clinical samples.

RESULTSThe nucleic acid positive rates of all 23 strains of measles virus are all 100% by the two methods. But to the detection of 18 clinical samples which are negative in measles isolation, the nest-RT-LAMP showed 56.52% positive rate of nucleic acid of measles virus and nest-RT-PCR showed 47.83%.

CONCLUSIONRT-LAMP is more sensitive than nest-RT-PCR.

Genome, Viral ; Humans ; Measles ; virology ; Measles virus ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVEA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HIV-1.

METHODSRT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The performance of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR(qRT-PCR).

RESULTSThe RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100%, respectively,in comparison with those of qRT-PCR.

CONCLUSIONRT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.

HIV-1 ; isolation & purification ; Naphthalenesulfonates ; Nucleic Acid Amplification Techniques ; methods ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Reverse Transcription ; Sensitivity and Specificity

A real-time RT-PCR (RT-qPCR) assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.
Animals ; Culicidae ; virology ; Encephalitis Virus, California ; isolation & purification ; Real-Time Polymerase Chain Reaction ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

This study was aimed to quantitatively analyze the mRNA level of bcr-abl fusion gene in K562/A02 cell line by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) technique. After being cultured for a period of time, the K562/A02 cell line was collected and RNA was extracted using TRIzoL kit. The real-time quantitative reverse transcriptase polymerase chain reaction technology was used to detect the level of bcr-abl fusion gene and internal reference abl gene. The results showed that a fine reproducibility was obtained between 10(7) and 10(3) copies/ml, reproducible sensitivity of RQ-RT-PCR was 10(-5). The expression of bcr-abl fusion gene in K562/A02 cells was higher and the level of bcr-abl mRNA was more than 100% in K562/A02 cells. It is concluded that RQ-RT-PCR is a reliable, sensitive and reproducible method for detecting mRNA level of bcr-abl fusion gene, which may be useful in monitoring the chronic myeloid leukemia.
Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity