Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition / 中华微生物学和免疫学杂志

Objective To obtain the full-length sequence of the vacuolar protein sorting 34 coding gene (vps34) of Sporothrix globosa (S. globosa) and to investigate the role of vps34 gene during the phase transition from mycelium to yeast in S. globosa. -ethods The 3′ end and 5′ end of the vps34 gene of S. globosa were amplified by rapid amplification of cDNA ends ( RACE ) . The obtained sequences were spliced and analyzed by bioinformatics software. Quantitative reverse transcription PCR ( qRT-PCR ) was used to analyze the expression of vps34 gene in mycelial and yeast phases. Results The vps34 gene of S. globosa was 3228 bp in length. The coding sequence was 3000 bp and encoded 999 amino acids with a mo-lecular mass of 111. 49×103 and an isoelectric point of 6. 38. It contained three domains including C2 PI3K class Ⅲ, PI3Ka Ⅲ and PI3Kc Ⅲ. The results of qRT-PCR showed that the expression of vps34 gene in yeast-phase S. globosa was higher than that in mycelial phase at 24 h (P<0. 05), and the greatest difference between them was observed at 48 h (P<0. 01). Conclusions Vps34p participates in the process of dimor-phic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the founda-tions for further study on the function of Vps34p.

Application of multiple MLL gene rearrangement detection techniques for children with acute mononuclear leukemia / 中华医学遗传学杂志

Objective@#To assess the value of detecting multiple rearrangements of MLL gene in children with acute mononuclear leukemia (AML).@*Methods@#Eighty six children with AML were analyzed by fluorescence in situ hybridization (FISH), chromosomal karyotyping and multiplex reverse transcription-PCR (RT-PCR).@*Results@#Cross signals were detected by FISH in 26 cases, and 30.2% were detected with MLL gene rearrangements. R-band karyotyping analysis revealed 14 translocations with breakages involving 11q23 and 5 other aberrations, which yielded an overall detection rate of 22.1%. Multiple RT-PCR has detected 12 fusion genes produced by the MLL translocation, which yielded a detection rate of 14.0%. A significant difference was found in the detection rate of the three methods (P<0.05).@*Conclusion@#Combined use of FISH, chromosomal karyotyping and multiplex RT-PCR can improve the detection of MLL gene rearrangements and provide important clues for clinical diagnosis, treatment and prognosis of AML.

Application of PCR-fluorescent probe in identifying genotypes of HCV / 中华临床感染病杂志

Objective To evaluate the application value of polymerase chain reaction (PCR)-fluorescence probe method in identifying genotypes of hepatitis C virus (HCV).Methods One hundred and sixty six serum samples from patients with chronic HCV infection were collected nationwide from March to June 2016.HCV Core-E1 gene region was amplified and sequenced by nested reverse transcription-PCR (RT nested-PCR)and genetic subtypes were analyzed by phylogenetic tree,meanwhile HCV genotypes were also determined by PCR-fluorescent probe method.Kappa test was used to compare the consistency of two methods.Results Among 166 samples detected by RT nested-PCR,the genotype of 66 samples (39.8%) was 1 b,34 (20.5%)was 2a,16 (9.6%)was 3a,27 was 3b (16.2%),23 (13.9%)was 6a.Two samples with 3b genotype detected by RT nested-PCR were identified as 1 b by PCR-fluorescent probe.The consistency rate of two methods was 98.7% (164 /166),there was no significant difference between two methods (χ2 =0.0492,P >0.05).Conclusion PCR-fluorescence probe method can accurately identify HCV genotypes and can be used in clinic.

Performance Evaluation of the PowerChek MERS (upE & ORF1a) Real-Time PCR Kit for the Detection of Middle East Respiratory Syndrome Coronavirus RNA

BACKGROUND: Molecular detection of Middle East respiratory syndrome coronavirus (MERS-CoV) using real-time reverse transcription (rRT)-PCR assays is the method of choice for diagnosis of MERS. We evaluated the performance of the PowerChek MERS (upE & ORF1a) real-time PCR Kit (PowerChek MERS assay; Kogene Biotech, Korea) a one-step rRT-PCR assay for the qualitative detection of MERS-CoV. METHODS: We evaluated PowerChek MERS assay performance in comparison with nested RT-PCR and sequencing of the RNA-dependent RNA polymerase (RdRp) and N genes. To evaluate diagnostic sensitivity and specificity, 100 clinical specimens (50 positive and 50 negative for MERS-CoV) were simultaneously tested by using the PowerChek MERS and sequencing assays. Assay performance, including limit of detection and precision, was evaluated in vitro by using MERS-CoV RNA transcripts. Analytical specificity was evaluated with a diverse collection of 16 respiratory virus–positive clinical specimens and 14 respiratory bacterial isolates. RESULTS: The 95% limits of detection of the PowerChek MERS assay for the upE and the open rading frame (ORF)1a were 16.2 copies/µL and 8.2 copies/µL, respectively. No cross-reactivity was observed. The diagnostic sensitivity and specificity of the PowerChek MERS assay were both 100% (95% confidence interval, 91.1–100%). CONCLUSIONS: The PowerChek MERS assay is a straightforward and accurate assay for detecting MERS-CoV RNA. The assay will be a useful tool for the rapid diagnosis of MERS and could prove especially important for MERS outbreak control.

Comparison of the Clinical Performance of Binax NOW RSV Versus Multiplex RT-PCR for Detection of Respiratory Syncytial Virus

BACKGROUND: Respiratory syncytial virus (RSV) is one of the most important causes of lower respiratory tract infection. The rapid antigen test is a simple, cheap, and quick method for RSV detection, however, it has an acknowledged low sensitivity. The aim of this study is to evaluate the diagnostic performance of the rapid antigen test by comparing it with a multiplex reverse transcription-PCR (RT-PCR). METHODS: A total of 557 nasopharyngeal aspirates or swabs that were submitted for both a rapid antigen test, Binax NOW RSV (Binax; Alere Scarborough, Inc., USA) and multiplex RT-PCR, Seeplex RV7 (Seegene Inc., Korea) were included in this study. We performed both tests according to the manufacturer's recommendations and analyzed the diagnostic performances of a rapid antigen tests based on the results of multiplex RT-PCR. RESULTS: Among the 557 specimens, the positive rates determined from the rapid antigen test and multiplex RT-PCR were 12.2% (N=68) and 25.1% (N=140), respectively. The relative sensitivity and specificity of the rapid antigen test were 46.4% and 99.3% based on the multiplex RT-PCR, respectively. Positive and negative predictive values were 95.6% and 84.7%, respectively. The diagnostic sensitivity was lower (28.6%) in children >36 months compared with children < or =36 months of age. Test sensitivity declined when RSV infection was accompanied by infection with other respiratory viruses. CONCLUSIONS: Binax NOW RSV exhibited good diagnostic performance, easy handling, and rapidity. However, it does have the possibility of false-negative results, and additional tests are needed when there is clinical suspicion of RSV infection.

Isolation and characterization of drought-responsive genes from peanut roots by suppression subtractive hybridization

Background Peanut (Arachis hypogaea L.) is an important economic and oilseed crop. Long-term rainless conditions and seasonal droughts can limit peanut yields and were conducive to preharvest aflatoxin contamination. To elucidate the molecular mechanisms by which peanut responds and adapts to water limited conditions, we isolated and characterized several drought-induced genes from peanut roots using a suppression subtractive hybridization (SSH) technique. Results RNA was extracted from peanut roots subjected to a water stress treatment (45% field capacity) and from control plants (75% field capacity), and used to generate an SSH cDNA library. A total of 111 non-redundant sequences were obtained, with 80 unique transcripts showing homology to known genes and 31 clones with no similarity to either hypothetical or known proteins. GO and KEGG analyses of these differentially expressed ESTs indicated that drought-related responses in peanut could mainly be attributed to genes involved in cellular structure and metabolism. In addition, we examined the expression patterns of seven differentially expressed candidate genes using real-time reverse transcription-PCR (qRT-PCR) and confirmed that all were up-regulated in roots in response to drought stress, but to differing extents. Conclusions We successfully constructed an SSH cDNA library in peanut roots and identified several drought-related genes. Our results serve as a foundation for future studies into the elucidation of the drought stress response mechanisms of peanut.

Effects of dihydroartemisinin on the expression level of Alpha-7 .3 giardin mRNA in C2 Giardia lamblia / 中国人兽共患病学报

Effects of dihydroartemisinin (DHA) on the expression level of Alpha-7 .3 giardin mRNA in C2 Giardia lam-blia was investigated in this study to explore the damage to skeleton protein of C 2 Giardia lamblia .Giardia lamblia was culti-vated respectively for 2 ,4 ,8 ,and 12 hours with modified TYI-S-33 medium containing 100 μg/mL and 200 μg/mL DHA , while the control group performed in the same experimental conditions without DHA .The expressive quantity of Alpha-7 .3 gi-ardin mRNA was determined by using real-time reverse transcription PCR ,and then we found that the expressive quantities of Alpha-7 .3 giardin mRNA with DHA were significantly lower than those in the control group .It’s suggested that dihydroarte-misinin has obvious inhibitory effect on the expression level of Alpha-7 .3 giardin mRNA in C2 Giardia lamblia .The actions of dihydroartemisinin on skeleton protein of C2 Giardia lamblia are effective .

Proliferation inhibition and apoptosis of human tongue cancer cell line Tca 8113 by andrographolide / 重庆医学

Objective To explore the effects of andrographolide(AD) on proliferation inhibition ,cells cycle distribution and the expression of Bax and Bcl‐2 mRNA in human tongue squamous cell carcinoma Tca8113 cells .Methods MTT was used to measure the levels of the proliferation of Tca8113 cells cultured with different concentraions of AD .Cell cycles were analyzed by flow cytom‐etry .RT‐polymerase chain reaction(RT‐PCR)technique was used to evaluate Bcl‐2 mRNA and Bax mRNA expression .Results An‐drographolide inhibited Tca8113 cells growth in a time and dose‐dependent manner ,the IC50 was 74 .66 μg/mL .A feature of apop‐tosis was observed under microscope .With a increase in concentration and increase in time ,the cells in G0/G1 phase increased from (39 .45 ± 0 .65)% to (63 .70 ± 0 .65)% ,the cells in S phase decreased from (56 .55 ± 0 .64)% to (32 .28 ± 0 .54)% .Statistics showed tha the cells arrested in G0/G1 phase .It was statistially significant in comparison with the control group (P< 0 .01) .AD could up‐regulate the experssion of Bax mRNA ,and AD could down‐regulate the experssion of Bcl‐2 mRNA .Conclusion An‐drographolide inhibited the growth of Tca8113 cells and increased the cell apoptosis .It may be promising as a new drug for treat‐ment of tongue squamous cell carcinoma .

A reverse transcription loop-mediated isothermal amplification assay to rapidly diagnose foot-and-mouth disease virus C

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to rapidly detect foot-and-mouth disease virus serotype C (FMDV C). By testing 10-fold serial dilutions of FMDV C samples, sensitivity of the FMDV C RT-LAMP was found to be 10 times higher than that of conventional reverse transcription-PCR (RT-PCR). No cross-reactivity with A, Asia 1, or O FMDV or swine vesicular disease virus (SVDV) indicated that FMDV C RT-LAMP may be an exciting novel method for detecting FMDV C.

Expression of specific genes of cardiomyocytes differentiated from human umbilical cord mesenchymal stem cells / 中国组织工程研究

BACKGROUND: Intramyocardial transplantation of autologous umbilical cord-derived mesenchymal stem cells for repair of myocardial tissue damage is paid increasing attention in the cardiovascular field. OBJECTIVE: Human umbilical cord mesenchymal stem cells were isolated and cultured. Passage 2 human umbilical cord mesenchymal stem cells were treated with various concentratins of 5-azacytidine (2.5, 5, 10, 20, 40, 80 μmol/L) for 24 hours . After removal of 5-azacytidine, cells were cultured for another 4 weeks. RESULTS AND CONCLUSION: Before 5-azacytidine treatment, filament-like structures or particles were not observed in the cells, but the amount of cytoplasm was less and uniform, nuclear/cytoplasm ratio was high, cells exhibited typical fusiform appearance and grew in a swirl-like manner, and nucleolus was obvious. After treatment with 5-azacytidine for 24 hours, some cells died in each group, and typical fusiform appearance turned into stick-like or column-like appearance, especial y in the 40 and 80 μmol/L 5-azacytidine groups. Reverse transcription-PCR results showed that atrial natriuretic peptide and α-skeletal actin gene expression levels were detected on human umbilical cord mesenchymal stem cells after treatment with 2.5 or 40 μmol/L 5-azacytidine for 4 weeks or with 5, 10, 20 μmol/L 5-azacytidine for 1, 2, 3 and 4 weeks. These findings suggest that 5-azacytidine-induced human umbilical cord mesenchymal stem cells express the specific gene of myocardiocytes.

Effects of SPARC on expression of TGF-βl and collagen type Ⅰ in keloid fibroblasts / 中华医学美学美容杂志

Objective To observe the effects of secreted protein,acidic and rich in cysteine (SPARC) on the expression of TGF-β1 and collagen type Ⅰ in cultured human keloid fibroblasts by real-time fluorescence quantitative RT-PCR.Methods In vitro keloid fibroblasts were stimulated by different concentrations of recombinant human SPARC,and with the control group for comparison,real-time fluorescence quantitative RT-PCR to detect expression of TGFβ1 and collagen type Ⅰ.Results Compared with the control group,the expression of TGF-β1 and collagen type Ⅰ was significantly increased in the experimental group.Conclusions SPARC could enhance the expression of TGF-β1 and collagen type Ⅰ in keloid fibroblasts significantly.

Application of real-time fluorescent RT-PCR for quantitative detection of SARS-Coronavirus / 临床检验杂志

Objective To establish a real-time fluorescent quantitative RT-PCR for detecting SARS-Coronavirus (CoV) mRNA in gargling liquid and serum of SARS patients.Methods The assay is based on simplified nested fluorescent RT-PCR. Total RNA was extracted from gargling liquid and serum by using high performance system and reverse-transcription by antisense primer.The specific TaqMan probe was designed according to the published DNA sequence of SARS-CoV polymerase and labeled with FAM and TAMRA. Standard curves for SARS-CoV quantification were prepared with serial dilutions of the recombinant plasmid.Results The method is highly sensitive and specific. The sensitivity of assay was 1?105 copies/L. The positive rate of SARS-CoV mRNA in the gargling liquid of patients was 65.0% (26/40) and 11.5% (6/52) in suspected patients, respectively. SARS-CoV mRNA was not detectable in the gargling liquid from 40 healthy individuals. The positive rate of SARS-CoV mRNA in the serum of SARS patients was 25.6% (10/39). PCR amplification products of 6 suspected patients with SARS-CoV mRNA were confirmed by sequencing and the sequence data were consistent with that of BJ01 AY278488.Conclusions Real-time fluorescent quantitative RT-PCR should be a rapid, specific tool for early diagnosis of SARS-CoV infection.

Reference genes selection in mRNA analysis of aging rat tissues / 基础医学与临床

Objective To investigate the proper inner control genes suitable for mRNA expression level comparison of aging rat tissues.Methods Real-time reverse transcription PCR was used to examine in aging rat tissues the expression level of G3pd(glyceraldehyde-3-phosphate dehydrogenase),ACTB(?-actin),H3f3b(H3 histone,family 3B),Arbp(acidic ribosomal phosphoprotein P0)and 18S(18S ribosomal RNA).Results The most stably expressed housekeeping gene in aging rat kidney was ACTB,in heart and lung G3pd showed the minimum variation;Arbp expression was the most stable one in different tissues.Conclusion For aging rat intra-tissue mRNA normalization at least two housekeeping genes should be used: one is the ribosomal RNA gene 18S and another one is Arbp.

Detection of Enterovirus in Cerebrospinal Fluid by Real-Time Nested Reverse Transcription Polymerase Chain Reaction / 대한진단검사의학회지

BACKGROUND: Enterovirus is a common cause of aseptic meningitis, respiratory disease and nonspecific febrile illness. The conventional methods for laboratory diagnosis of enterovirus infections have been virus culture and serotyping by an immunofluorecent test. We studied a new and more rapid approach for enterovirus detection in cerebrospinal fluid (CSF) by real-time nested PCR. METHODS: This study was performed on 50 CSF specimens from patients suspected of aseptic meningitis. Enterovirus was detected in CSF by PCRs for 3 different targets and real-time nested PCR. Enterovirus culture was also performed in 44 CSF specimens. RESULTS: The positive rate of PCRs for each of the 3 different targets was 26.0%, 40.0%, or 46.0%, and that of real-time nested PCR was 86.0%. Only 6.8% were positive in culture. Thus, the positive rate of real-time nested PCR was much higher than other methods. CONCLUSIONS: Our study revealed that the real-time nested PCR should be useful for diagnosis of enterovirus infections because of a high sensitivity and rapid detection.

Cloning of L-Methionine ?-lyase gene / 医学研究生学报

Objective:To identify,clone,sequence the L-Methionine ?-lyase gene from Trichomonas vaginalis(TVMGL1).Methods:Total RNA was prepared from primitive protozoon trichomonas vaginalis,cDNA fragment encoding Methionine ?-lyase gene was amplified by RT-PCR using specific primers,and then was cloned into pGEM-T vector.Inserted Methionine ?-lyase gene was sequenced by ABI 3730 DNA Sequencer.Results:Analysis indicates that the DNA fragment is 1 191 base pairs in length and has high nucleotide homology with that reported previously.Conclusion:L-Methionine ?-lyase gene was successfully cloned.

Comparison of the reverse transcription-PCR with the branched DNA assay for measurement of human immunodeficiency virus type 1 RNA levels in plasma of Korean patients

Viral load testing of human immunodeficiency virus (HIV) is an essential tool for initiating and monitoring the antiretroviral therapy for HIV patients. To this end, several methods including polymerase chain reaction (PCR), branched DNA (bDNA), nucleic acid sequence based amplification assay (NASBA) and internally controlled virion PCR (ICV PCR) have become available. Of these methods, the standard reverse transcription-PCR (RT-PCR) assay has been widely used in Korea. However, no comparison study has been performed among the various detection methods currently used in Korean patients. We evaluated the correlation and agreement between the PCR and the branched DNA (bDNA) assay for measurement of HIV RNA in Korean patients. Eighty randomly selected samples from HIV-1-seropositive patients visiting Yonsei Medical Center Severance Hospital were studied. We found that these assays show good agreement, have a reliable correlation (r = 0.92, mean difference in log10 copies/ mL +/- 2 standard deviation = 0.098 +/- 0.805) and produce values whose relationship is given by the following equation: log10v3 bDNA = -0.3405 + 1.0601 x log10RT-PCR. Thus, we conclude that these two methods may allow direct comparison of the results obtained from different assay systems.

Expression of heat shock protein 90? in colon carcinoma tissues and cells and its relationship to chemoresistance / 中国普通外科杂志

Objective To explore the expression of heat shock protein 90?(HSP90?)in colon carcinoma tissues and HCT-8 cell line,and to investigate the relationship of HCT-8 cells to chemoresistance.Methods The expression of HSP90 ? in colon carcinoma tissues was detected by immunohistochemistry and the relationship of HSP90? expression with the Dukes stage and pathological grade was analyzed,The medium effective concentration(IC50)of vincristine(VCR)to colon cancer parent cells HCT-8 and drug resistance cells HCT-8/VCR was detected by MTT method.The expression difference of HSP90?mRNA in of HCT-8 cells and HCT-8/VCR cells was detected by RT-PCR to investigate its relationship to the chemoresistance.Results The positive expression rate of HSP90 ? was higher in colon carcinoma tissues(41.67%)than non-cancerous colon tissues(16.67%)(P0.05).The drug resistance of HCT-8/VCR cell line to VCR was significantly higher than that of HCT-8 cell line(P

Development of Quantitative Reverse Transcription-Polymerase Chain Reaction for the Mesurement of Angiotensin Converting Enzyme mRNA

BACKGROUND: The analysis of ACE gene expression in vital to study the role of angiotensin conveting enzyme(ACE) in the pathogenesis of cardiovascular disease. Traditionally, levels of individual mRNA expression have been analyzed by semiquantitative Northern blotting, which requires a large quantity of tissue. Therefore, gene expression of a little biopsy specimen from the human heart or atherectomy specimen from the blood vessel cannot be measured easily. Reverse transcription-polymerase chain reaction(RT-PCR) is very effective, sensitive and rapid method of detecting the method of quantitative RT-PCR(QRT-PCR) using recombinant RNA template as internal standard to measure the expression of ACE. METHOD: Recombinant RNA(rcRNA) was designed to yield PCR product which differs in size by about 200bp from that of the target RNA. Initially, spacer gene, which was composed of ACE sense primer, antisense primer, T7 promotor and poly(dT) tail with glutathione transferase(GSTM) gene of 180bp in the middle, was constructed. Then, standard rcRNA was obtained by in vitro transcription. Target RNA was mixed with rcRNA and amplified by PCR, togather with P-dCTP. PCR products were analyzed by gel electrophoresis. For quantitation, either gel was cut and radioactivity was counted or gel was dried and exposed to X-ray film and density was measured using image densitometer. We carried out semiquantitative RT-PCR to study the modulation of ACE expression in vascular smooth muscle cell(VSMC) by dexamethasone and basis FGF(bFGF). RESULT: The size difference of PCR products from the standard RNA and the extracted target RNA was matched as designed. By using QRT-PCR, there was 1.7*10(8) ACE mRNA molecules in 1 ng of rat lung total RNA. bFGF and dexamethasone upregulated ACE mRNA expression in cultured VSMC. CONCLUSION: These results suggest that RT-PCR using rcRNA as internal standard is a very useful method for quantitation or semiquantitation of ACE mRNA from a small amount of tissue or cultured cells. Expression of ACE in VSMC can be modulated by various stimuli such as basic FGF and dexamethasone. QRT-PCR could be widely used in the studies of expression of specific human genes.