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

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

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

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

Molecular detection of enterovirus (EV)71 RNA based on PCR methods is a quick and sensitive approach. At present, different PCR-based methods for EV71 RNA detection are available, but comparisons of results obtained using different approaches are limited. This study is to compare the analytical sensitivity and specificity of different real-time reverse transcription-polymerase chain reaction (rRT-PCR) and conventional reverse transcription-polymerase chain reaction (cRT-PCR) assays for enterovirus and EV71 detection, Altogether, three rRT-PCR assays and one cRT-PCR assay targeting the 5'UTR gene for universal detection of enterovirus; two rRT-PCR assays andone cRT-PCR assay targeting the VP1 gene for specific detection of EV 71 were examined. All assays showed good specificity. The detection sensitivity ranged from 8.19 x 10 to 8.19 x 10(5) copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All rRT-PCR assays showed 100% sensitivity for clinical specimens.
Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Enterovirus Infections ; diagnosis ; virology ; Humans ; Real-Time Polymerase Chain Reaction ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Child ; Child, Preschool ; Computer Systems ; Humans ; Phylogeny ; Polymerase Chain Reaction ; methods ; Respiratory Syncytial Viruses ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods

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

OBJECTIVETo detect tumor specific chromosome translocations and associated fusion transcripts in paraffin-embedded tissue by interphase fluorescence in-situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR), respectively, and to evaluate their diagnostic values for Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET).

METHODSNuclei of the tumor cells and total RNA were extracted from 10 cases of ES/PNET. Interphase FISH was utilized to analyze the EWS gene translocation with a dual color, break apart probe (Vysis company). RT-PCR was used to detect t (11; 22) (q24; q12) and t (21; 22) (q22; q12) fusion transcripts.

RESULTSAmong 10 cases of ES/PNET, the EWS-FLI1 fusion transcript was detected in 8 by RT-PCR. EWS-ERG fusion transcript was not detected in any of the cases. EWS gene translocation was found in 9 of 10 cases by FISH.

CONCLUSIONSInterphase FISH and RT-PCR can be reliably applied to paraffin-embedded tissues for molecular diagnosis of ES/PNET. Between the two approaches, interphase FISH provides a more sensitive and stable result.

Adolescent ; Adult ; Child ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Neuroectodermal Tumors, Primitive, Peripheral ; diagnosis ; genetics ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sarcoma, Ewing ; diagnosis ; genetics

OBJECTIVEUsing polymerase chain reaction-reverse blot dot (PCR-RDB) technique to establish a new method for hepatitis C virus (HCV) genotyping and to study the distribution of HCV genotypes in Foshan area.

METHODSHCV primers and probes were designed in 5'-untranslated region (nt-1-nt-299) of HCV. HCV RNA in serum was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin-labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype 1a to 3b) on Biodyne C membrane to genotype HCV by color development while adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluorescent quantitative (FQ)-PCR. 60 FQ-PCR-positive HCV sera from Foshan area were genotyped using this assay.

RESULTSAll 60 sera could be successfully genotyped by PCR-RBD. 50 (83.3%) cases were found to be genotype 1b, 2 (3.3%) as genotype 1a and 2 (3.3%) as genotype 2a while 5 (8.0%) to be mixture of genotype 1a and 1b, and 1 (1.7%) to be mixture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. The results of PCR-RDB genotyping methods coincided with sequence analysis.

CONCLUSIONNewly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies. The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area.

Genotype ; Hepacivirus ; classification ; genetics ; Hepatitis C ; virology ; Humans ; Immunoblotting ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity