ABSTRACT
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.
Subject(s)
Coronavirus Infections , Diagnosis , In Vitro Techniques , Limit of Detection , Methods , Middle East Respiratory Syndrome Coronavirus , Middle East , Real-Time Polymerase Chain Reaction , Reverse Transcription , RNA , RNA-Dependent RNA Polymerase , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Arachis/genetics , Stress, Physiological/genetics , Droughts , RNA/isolation & purification , Gene Library , Sequence Analysis , DNA, Complementary/isolation & purification , Plant Roots , Gene Expression Regulation, Plant , Reverse Transcriptase Polymerase Chain Reaction , Dehydration , Nucleic Acid Hybridization/methodsABSTRACT
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 .
ABSTRACT
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.