The Department of Microbiology, Parasitology, and Biotechnology is delighted to announce to the scientific community and extend its congratulations to Dr. Emmanuel George Kifaro, a member of our academic staff, on the successful defense of his PhD on 6th September 2023 at Sokoine University of Agriculture (SUA). His PhD thesis is titled “Microparticles for Direct Detection of Viral RNA from Non-Invasive Matrices and Extensive Multiplexing of Respiratory Viruses.”
Dr. Emmanuel George Kifaro began his PhD program at the Department of Microbiology, Parasitology, and Biotechnology within the College of Veterinary Medicine and Biomedical Sciences (CVMBS) in October 2018. Employed by Sokoine University of Agriculture, Dr. Kifaro serves as an Assistant Lecturer in Molecular Biology and Biotechnology within the same department. He was guided and supervised by Professor Gerald Misinzo from CVMBS, SUA, and Professor Sang Kyung Kim from the Korea Institute of Science and Technology (KIST). Dr. Kifaro’s primary research focuses on Molecular Biology and Biotechnology.
List of members of the panel for the viva voce examination
| 1 | Chairperson | Prof. Claudius D. Luziga |
| 2 | Appointee of the Principal, CVMBS | Dr. Isaac Kashoma |
| 3 | Appointee of the Head of Department | Dr. Jahashi Nzalawahe |
| 4 | Independent Internal Examiner 1 who did not supervise | Prof. Christopher J. Kasanga |
| 5 | Independent Internal Examiner 2 who did not supervise | Dr. Elisa Mwega |
Summary of findings
The advent of the polymerase chain reaction (PCR) has revolutionized the field of viral diagnostics. Today, PCR is globally recognized as the gold standard for diagnosing and monitoring viral infectious diseases in both humans and animals. Its heightened sensitivity and reliability make it an unparalleled tool for nucleic acid detection across diverse sample types. Nevertheless, the intricate infrastructure and technicalities surrounding PCR limit its use in field diagnostics. Furthermore, the complexity of monitoring numerous detection dyes from individual excitation sources constrains the broad-scale detection of multiple targets. Non-invasive matrices like saliva and stool, which are cost-effective and easily collected, often pose challenges for molecular pathogen detection due to their high content of substances inhibitory to PCR.
This research honed in on the design and utility of hydrogel microparticles, specifically the primer immobilized network (PIN) and its thermal-responsive counterpart, tPIN. These were employed for recognizing and distinguishing RNA viruses. Our initial findings demonstrated the viability of PIN particles for executing direct quantitative reverse transcription PCR (dirRT-qPCR) on viral RNA extracted from saliva, circumventing RNA purification. We further evidenced that these PIN particles could transport viral RNA from stool samples for subsequent real-time PCR (qPCR) analysis, again without the necessity for RNA purification. Lastly, we unveiled the potential of tPIN particles, encapsulated within low melting point agarose gel (LMPA), to facilitate multiplex one-step RT-qPCR for prevalent respiratory viruses.
A range of physical and chemical attributes of the PIN particles played a pivotal role in facilitating dirRT-qPCR of viral RNA from saliva. These particles adeptly filtered micro-volumes of the solution, enabling effective RNA priming and conversion to complementary DNA (cDNA) using the embedded RT primers. Following a swift heat treatment for viral lysis, the assay pinpointed the matrix (M) gene for influenza A virus (IAV) and the 5’ untranslated region (5’ UTR) for chicken coronavirus (CoV) in saliva samples with remarkable efficiency. By introducing the reverse transcriptase enzyme and diluting the matrix, the assay’s sensitivity was amplified. Furthermore, harmonizing the PIN with microfluidic PCR technology slashed the processing time dramatically.
For stool samples mixed with the viral solution, a brief heat treatment was applied to initiate viral lysis, liberating viral RNA amidst other components and neutralizing PCR inhibitors sensitive to heat. Captured viral RNA was subsequently converted to a pool of cDNA. This assay outperformed many commercial viral RNA purification kits in terms of amplification efficiency and detection limits.
The multiplex qPCR approach is crucial in the medical realm for swift differential diagnosis, especially when multiple infections are in play. Herein, we introduced a one-step RT-qPCR assay, leveraging tPINs, to detect and differentiate 12 key respiratory viruses. These tPINs, functioning as distinct reaction chambers on a tailored qPCR chip, ensured an independent and target-specific amplification. By prioritizing Influenza A and B viruses and incorporating ten other respiratory viruses, this technique yielded exceptional amplification efficiencies.
In summary, hydrogel PIN technologies are poised to make a significant impact on on-the-spot diagnostics, enhancing disease monitoring using complex non-invasive matrices. Concurrently, tPINs herald a new era for concurrent pathogen detection on singular microfluidic qPCR chips, accelerating differential diagnoses via ultrafast one-step RT-qPCR platforms.
Publications:
- Kifaro, E. G., Kim, M. J., Jung, S., Noh, J. Y., Song, C. S., Misinzo, G., & Kim, S. K. (2022). Direct Reverse Transcription Real-Time PCR of Viral RNA from Saliva Samples Using Hydrogel Microparticles. Biochip J, 16(4), 409-421. FULL TEXT
- Kifaro, E. G., Kim, M. J., Jung, S., Jang, Y. H., Moon, S., Lee, D. H., Song, C. S., Misinzo, G., & Kim, S. K. (2023). Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance. Diagnostics (Basel), 13(2). FULL TEXT
“Let’s all join hands in congratulating him”
Augustino Alfred Chengula, PhD
Head of Department of Microbiology Parasitology and Biotechnology
The College of Veterinary Medicine and Biomedical Sciences, Sokoine University of Agriculture