Ebola virus is a deadly causative agent with a high mortality rate of up to 90%, therefore it has been classified by the Center for Disease Control and Prevention (CDC) as a category A biological agent. The World Health Organization (WHO) recommended using RT-PCR based assays to rapidly detect the virus. In the present study, we established an in-house assay for detection of Zaire ebolavirus via real-time RT-PCR. The nucleotide sequence of the Zaire ebolavirus nucleoprotein (NP) gene was retrieved from the Genbank for designing primer pairs and probes using Primer Express 3.0 software. The RNA positive control was generated by in vitro RNA transcript synthesis. The optimal components in the 20 μl final volume of the real-time RT-PCR assay were 10 μl 2X QuantiTectProbeRT-PCR master mix, 0,6 μM of each primer, 0,1 μM of the probe, 0,2 μl RT mix and 5 μl of RNA template. The thermal cycle conditions were as follows: 50oC for 30 min, 95°C for 15 min, then 45 cycles of 15 s at 94°C, 60s at 60°C. The limit of detection of the assay was 100 copies/reaction and 1414 FFU/ml with the positive RNA panel and sample panel of RNA extracted from cell culture supernatants of cells infected with Zaire ebolavirus 2014/Gueckedou-C05, respectively. The specificity of this assay was 100% when tested with the positive RNA panel of Ebola virus and other haemorrhagic fever viruses. In conclusion, we successfully established an in-house real-time RT-PCR assay for detection of Zaire ebolavirus in Vietnam with a limit of detection of 1414 FFU/ml and specificity of 100%.

In Vietnam, nasopharyngeal carcinoma (NPC) is the eighth most common cause of death from cancer. Cell-free Epstein Barr virus DNA (cf-EBV DNA) was reported to be present in almost all NPC patients. However, currently available assays in Vietnam can detect cf-EBV DNA in only 67.6% of NPC patients, thus leaving 32.4% of cancer cases undetected. Therefore, in this study, we aim to develop a highly sensitive quantitative PCR (qPCR) assay that measures the load of cf-EBV DNA for the purpose of early detection of NPC, and then evaluate the sensitivity and the specificity of the developed qPCR assay on the clinical samples. The major methods used in this study include primer/TaqMan probe design, cf-DNA extraction, optimization of qPCR assay and statistical analysis. Using an international standard panel from the Chinese University of HongKong, the linear range of developed qPCR assay is from 50-150,000 copies/ml (R2 = 0.99613) and the detection limit has been shown to be 25 copies/ml. The developed assay could detect cf-EBV DNA with a sensitivity of 96.9% (31/32 NPC patients) and cf-EBV DNA has not been detected in 103 out of 105 healthy controls, which corresponds to a specificity of 98%. Consequently, the performance of the optimal assay has achieved remarkably high sensitivity and specificity. Moreover, the detection limit of our optimal qPCR assay is 25 copies/ml of plasma, which is at least ten times better than other assays tested in recent studies in Vietnam. This developed qPCR assay will also form the basis for further studies in Vietnam and will open many new applications in management of NPC.

An in vitro transcribed RNA protocol of 5 pathogenic Ebola virus species ZEBOV, SEBOV, BEBOV, TEBOV, REBOV using synthesized plasmids was established with high efficient and following steps: (1) Cloning synthesized plasmids containing NP sequence and 3'UTR region of EBOV in E. coli; (2) 20 μl volume in vitro RNA transcription reaction: 5X TranscriptAid Reaction Buffer; 10 mM dNTPs mix; 0.8 - 1 μg plasmid DNA; 2 μl TranscriptAid Enzyme Mix and DEPC-treated water, 37 °C /2 hours, 2 μl DNase I, 37 °C /15 minutes by TranscriptAid T7 High Yield Transcription Kit. (3) Evaluation of in vitro transcribed RNA by electrophoresis and one-step RT-PCR assay. The in vitro transcribed RNA of 5 Ebola virus species with 900-1400 ng/µl concentration and from 1.8-2.1 A260/A280 ratio.

Objectives: To determine the changes in serum HBV RNA levels in treatment-naïve chronic hepatitis B (CHB) patients who were treated with Tenofovir Disoproxil Fumarate (TDF). Methods: 77 treatment-naïve CHB patients were treated with long-term TDF monotherapy at the Department of Infectious Diseases, Military Hospital 103, Vietnam Military Medical University from 2017 to 2020. Samples were collected at several time points: At the baseline, after 3, 6, 9, and 12 months of TDF treatment. Serum HBV DNA and HBV RNA levels were quantified by the Real Time RT-PCR method. Statistical analyses were performed with Medcalc 20.019. Results: Serum HBV RNA levels tended to decrease during the TDF treatment in a biphasic pattern. In the first phase, from baseline to 3 months of treatment, HBV RNA levels decreased rapidly (the median slope of the decrease was 0.38 log copies/mL/month). In the second phase, from 3 - 12 months of treatment, serum HBV RNA levels decreased more slowly than in the first phase (the median slope of the decrease was 0.09 log copies/mL/month; p < 0.05). Serum HBV RNA levels decreased more slowly than serum HBV DNA levels in the first phase, but there was no significant difference in the second phase (p > 0.05). Conclusion: Serum HBV RNA levels decreased in a biphasic pattern with a different slope during TDF treatment. Serum HBV RNA levels decreased more slowly than HBV DNA and may complement this marker in the assessment of treatment outcomes and prognosis for chronic hepatitis B.