The Effect of RNA Storage on Dengue Virus Detection Capability Using Reverse Transcription PCR and Reverse Transcription LAMP Methods
Pengaruh Penyimpanan RNA Terhadap Kemampuan Deteksi Virus Dengue Menggunakan Metode Reverse Transcription PCR dan Reverse Transcription LAMP
DOI:
https://doi.org/10.21070/ups.11383Keywords:
Dengue virus, conventional RT-PCR, , RT-LAMP, sample storage duration, RNA degradationAbstract
Dengue Hemorrhagic Fever remains a significant public health concern, with East Java Province recording an incidence rate of 78.83 per 100,000 population in 2024. This study analyzed the ability to detect dengue virus in samples stored for ≥72 hours using conventional RT-PCR and RT-LAMP. A comparative laboratory design was applied to 30 whole blood samples from patients with suspected dengue, stored at 0–4°C across six storage durations (72–192 hours). RNA was extracted using the spin-column method, then examined using conventional RT-PCR (C–prM gene, 511 bp) and RT-LAMP (six primers, read through colorimetric change). RT-LAMP yielded positive results in 26 samples (86.7%), whereas conventional RT-PCR detected only 1 sample (3.3%). The Fisher–Freeman–Halton test showed no significant association between storage duration and RT-LAMP results (p=0.660), while the McNemar test showed a significant difference between RT-PCR and RT-LAMP outcomes (p<0.001). RT-LAMP shows potential as an alternative when RNA quality declines due to storage.
Downloads
References
WHO, "Dengue and severe dengue." Accessed: Jun. 30, 2026. [Online]. Available: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue
Z. Hussain, S. Batool, A. U. Khan, R. Shaheen, M. Asif, and A. Ghulam, "Comparative Evaluation of ELISA, PCR, and NS1 Antigen Detection for Dengue Virus Diagnosis," Insights-Journal of Health and Rehabilitation, vol. 3, no. 1, pp. 574–580, 2025, doi: 10.71000/r4b32w02.
D. Anggraini and M. Ariyanti, "Aspek Klinis dan Pemeriksaan Laboratorium untuk Diagnosis Demam Berdarah Dengue," Scientific Journal, vol. 1, no. 1, pp. 70–78, 2022, doi: 10.56260/sciena.v1i1.13.
G. Iqbal, H. Javed, F. A. Raza, U. F. Gohar, W. Fatima, and M. Khurshid, "Diagnosis of Acute Dengue Virus Infection Using Enzyme-Linked Immunosorbent Assay and Real-Time PCR," Canadian Journal of Infectious Diseases and Medical Microbiology, vol. 2023, pp. 1–8, 2023, doi: 10.1155/2023/3995366.
A. R. Siregar, T. Wibawa, and N. Wijayanti, "Early Detection and Serotyping of Dengue Viruses by Using Reverse Transcription Polymerase Chain Reaction (RT-PCR) 2 Primers," Indonesian Journal of Biotechnology, vol. 16, no. 2, pp. 71–75, 2011, doi: 10.22146/ijbiotech.7836.
G. A. Santiago et al., "Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus," PLoS Neglected Tropical Diseases, vol. 7, no. 7, pp. e2311–e2311, 2013, doi: 10.1371/journal.pntd.0002311.
S. Aziz, R. C. Sari, D. Priyanto, and T. Ramadhani, "Design and Evaluation of Degenerate Primers Targeting the NS3 Gene for Detection of Dengue Virus by RT-PCR," Acta Biochimica Indonesiana, vol. 8, no. 1, pp. 190–198, 2025, doi: 10.32889/actabioina.190.
S. Kumar, S. Sharma, N. Bhardwaj, V. Pande, D. Savargaonkar, and A. R. Anvikar, "Advanced Lyophilised Loop Me-diated Isothermal Amplification (L-LAMP) Based Point of Care Technique for the Detection of Dengue Virus," Journal of Virological Methods, vol. 293, pp. 114168–114168, 2021, doi: 10.1016/j.jviromet.2021.114168.
L. Hurtado-Gómez, M. A. Castro-Jiménez, J. D. Sepúlveda-Arias, J. A. Cardona-Ospina, and A. J. Rodriguez-Morales, "Development and Validation of a Combined RT-LAMP Assay for the Rapid and Sensitive Detection of Dengue Vi-rus in Clinical Samples from Colombia," Diagnostics, vol. 15, no. 5, pp. 570–570, 2025, doi: 10.3390/diagnostics15050570.
J. G. Kim, S. Yun, Y. Kim, S. Lee, and H. Kim, "Rapid Discriminative Detection of Dengue Viruses via Loop-Mediated Isothermal Amplification," Talanta, vol. 190, pp. 391–396, 2018, doi: 10.1016/j.talanta.2018.08.019.
K. Rani, S. Sharma, A. Gupta, R. Verma, and P. Singh, "Dengue Virus: A Global Concern and Advances in Treatment Strategies," Microbe, vol. 9, pp. 100591–100591, 2025, doi: 10.1016/j.microb.2025.100591.
S. C. Kaushik, S. Singh, and P. Srivastava, "Detection of Dengue Virus in Human Blood Samples from Selected Lo-calities of Delhi and NCR," Journal of Communicable Diseases, vol. 51, no. 3, pp. 10–14, 2019, doi: 10.24321/0019.5138.201920.
A. A. Rogers, R. E. Baumann, G. A. Borillo, R. M. Kagan, H. J. Batterman, M. M. Galdzicka, and E. M. Marlowe, "Evaluation of transport media and specimen transport conditions for the detection of SARS-CoV-2 by use of real-time reverse transcription-PCR," Journal of Clinical Microbiology, vol. 58, no. 8, pp. e00708-20–e00708-20, 2020, doi: 10.1128/JCM.00708-20.
S. A. Bustin and T. Nolan, "Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction," Jour-nal of Biomolecular Techniques, vol. 15, no. 3, pp. 155–166, 2004.
B. La Scola and D. Raoult, "Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-year experience (1993 to 1998)," Journal of Clinical Microbiology, vol. 37, no. 6, pp. 1899–1905, 1999, doi: 10.1128/JCM.37.6.1899-1905.1999.
M. Parida et al., "Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay," Journal of Clinical Microbiology, vol. 43, no. 6, pp. 2895–2903, 2005, doi: 10.1128/JCM.43.6.2895-2903.2005.
L.-H. Huang, P.-H. Lin, K.-W. Tsai, L.-J. Wang, Y.-H. Huang, H.-C. Kuo, and S.-C. Li, "The effects of storage temper-ature and duration of blood samples on DNA and RNA qualities," PLoS ONE, vol. 12, no. 9, pp. e0184692–e0184692, 2017, doi: 10.1371/journal.pone.0184692.
K. Bonner, R. A. Siemieniuk, A. Boozary, T. Roberts, E. Fajardo, and J. Cohn, "Expanding access to HIV viral load testing: A systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresh-olds," PLoS ONE, vol. 9, no. 12, pp. e113813–e113813, 2014, doi: 10.1371/journal.pone.0113813.
Kementerian Kesehatan Republik Indonesia, Profil Kesehatan Indonesia 2024. Jakarta: Kementerian Kesehatan RI, 2025, hal. 205.
B.-T. Teoh, S.-S. Sam, K.-K. Tan, M. B. Danlami, M.-H. Shu, J. Johari, P.-S. Hooi, D. Brooks, O. Piepenburg, O. Nentwich, A. Wilder-Smith, L. Franco, A. Tenorio, and S. AbuBakar, "Early detection of dengue virus by use of re-verse transcription-recombinase polymerase amplification," Journal of Clinical Microbiology, vol. 53, no. 3, pp. 830–837, 2015, doi: 10.1128/JCM.02648-14.
H.-J. Burkardt, "Standardization and quality control of PCR analyses," Clinical Chemistry and Laboratory Medicine, vol. 38, no. 2, pp. 87–91, 2000, doi: 10.1515/CCLM.2000.014.
C. J. Morgan, "Use of proper statistical techniques for research studies with small samples," American Journal of Physiology-Lung Cellular and Molecular Physiology, vol. 313, no. 5, pp. L873– L877, 2017, doi: 10.1152/ajplung.00238.2017.
S. F. Ahamed, R. Vivek, S. Kotabagi, K. Nayak, A. Chandele, K. Murali-Krishna, and A. Shet, "Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India," Journal of Virological Methods, vol. 244, pp. 46–54, 2017, doi: 10.1016/j.jviromet.2017.02.014.
T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase, "Loop- mediated isothermal amplification of DNA," Nucleic Acids Research, vol. 28, no. 12, p. e63, 2000, doi: 10.1093/nar/28.12.e63.
S. Fleige and M. W. Pfaffl, "RNA integrity and the effect on the real-time qRT-PCR performance," Molecular Aspects of Medicine, vol. 27, no. 2–3, pp. 126–139, 2006, doi: 10.1016/j.mam.2005.12.003.
Q. Huang, L. Baum, and W.-L. Fu, "Simple and practical staining of DNA with GelRed in agarose gel electrophoresis," Clinical Laboratory, vol. 56, no. 3–4, pp. 149–152, 2010.
I. Prado, D. Rosario, L. Bernardo, M. Álvarez, R. Rodríguez, S. Vázquez, and M. G. Guzmán, "PCR detection of dengue virus using dried whole blood spotted on filter paper," Journal of Virological Methods, vol. 125, no. 1, pp. 75–81, 2005, doi: 10.1016/j.jviromet.2005.01.001.
Downloads
Additional Files
Posted
Categories
License
Copyright (c) 2026 UMSIDA Preprints Server
This work is licensed under a Creative Commons Attribution 4.0 International License.
