Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel

  • Dyah Widiastuti Balai Penelitian dan Pengembangan Kesehatan Banjarnegara, Jalan Selamanik Nomor 16 A Banjarnegara, Jawa Tengah, Indonesia
  • Agustiningsih Agustiningsih Pusat Penelitian dan Pengembangan Biomedis dan Teknologi Dasar Kesehatan, Jalan Percetakan Negara Nomor 23 Jakarta Pusat, Indonesia
  • Ihda Zuyina Ratna Sari Balai Penelitian dan Pengembangan Kesehatan Banjarnegara, Jalan Selamanik Nomor 16 A Banjarnegara, Jawa Tengah, Indonesia
  • Tri Ramadhani Balai Penelitian dan Pengembangan Kesehatan Banjarnegara, Jalan Selamanik Nomor 16 A Banjarnegara, Jawa Tengah, Indonesia
Keywords: V1016G, Aedes aegypti, melting curve, allele specific polymerase chain

Abstract

Detection of V1016G mutation is important for identifying the mechanism of  synthetic pyrethroid resistance in Aedes aegypti population. The previous method has described an allele specific polymerase chain reaction (AS-PCR) using conventional PCR to detect the mutation. Although the method has great differentiating power and reproducibility, faster and more sensitive genotyping method is essential to accurately detect the mutation. This study evaluate the used of SYBR® Green real-time PCR and melting curve analysis (MCA) to identify the V1016G mutation. The collection of homozygous 1016G, heterozygous, and wild type (1016 V) mosquitoes DNA genome was extracted using genomic DNA mini kit. The SsoAdvanced™ Universal SYBR® Green Supermix was used to identify alleles by real-time PCR followed melting curve analysis of the amplicons. Melting curve analysis produced reproducible results for the loci tested. The melting temperature was reached at 78.5 oC for homozygous 1016G mosquito and at 86 oC for wild type mosquito. Meanwhile, the heterozigous mosquito revealed two peaks of melting temperature at both 78.5 oC and 86 oC. These easily interpretable and distinguishable melting curve results were consistent with AS-PCR results obtained for the same alleles. The described MCA application for screening V1016G mutation is fast and widely accessible also could be implemented under field conditions

References

1. Sunaryo S, Widiastuti D. Resistensi Aedes aegypti terhadap insektisida kelompok organopospat dan sintetik piretroid di Provinsi Sumatera Utara dan Provinsi Jambi. BALABA. 2018; 14(1):95-106.doi:10.22435/blb.v14i1.304.

2. Amelia-Yap ZH, Chen CD, Sofian-Azirun M, Low VL. Pyrethroid resistance in the dengue vector Aedes aegypti in Southeast Asia: present situation and prospects for management. Parasit Vectors. 2018;11(1):1-17. doi:10.1186/s13071-018-2899-0.

3. Badolo A, Sombié A, Pignatelli PM, Sanon A, Yaméogo F, Wangrawa DW, et al. Insecticide resistance levels and mechanisms in Aedes aegypti populations in and around Ouagadougou, Burkina Faso. PLoS Negl Trop Dis. 2019;13(5):1-17. doi: 10.1371/journal.pntd.0007439.

4. Amelia-Yap ZH, Sofian-Azirun M, Chen CD, Lau KW, Suana IW, Syahputra E, et al. V1016G point mutation: the key mutation in the voltage-gated sodium channel (VGSC) gene of pyrethroid-resistant Aedes aegypti (Diptera: Culicidae) in Indonesia. J Med Entomol. 2019;56(4):953-8. doi:10.1093/jme/tjz035.

5. Chen M, Du Y, Wu S, Nomura Y, Zhu G, Zhorov BS, et al. Molecular evidence of sequential evolution of DDT-and pyrethroid-resistant sodium channel in Aedes aegypti. PLoS Negl Trop Dis. 2019;13(6):1-21. doi:10.1371/journal.pntd.0007432.


6. Fernando SD, Hapugoda M, Perera R, Saavedra-Rodriguez K, Black WC, De Silva NK. First report of V1016G and S989P knockdown resistant (kdr) mutations in pyrethroid-resistant Sri Lankan Aedes aegypti mosquitoes. Parasites Vectors. 2018;11:1-6. doi:10.1186/s13071-018-3113-0.

7. Ariati J, Perwitasari D, Marina R, Shinta S, Lasut D, Nusa R, et al. Status kerentanan Aedes aegypti terhadap insektisida golongan organofosfat dan piretroid di Indonesia. J Ekol Kesehat. 2019;17(3):135-45. doi: 10.22435/jek.17.3.847.135-145.

8. Seixas G, Grigoraki L, Weetman D, Vicente JL, Silva AC, Pinto J, et al. Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal). PLoS Negl Trop Dis. 2017;11(7):1-16. doi:10.1371/journal.pntd.0005799.

9. Wuliandari JR, Hoffmann AA, Tantowijoyo W, Endersby-Harshman NM. Frequency of kdr mutations in the voltage-sensitive sodium channel (VSSC) gene in Aedes aegypti from Yogyakarta and implications for Wolbachia-infected mosquito trials. Parasites & Vectors. 2020;13:1-15. doi:10.1186/s13071-020-04304-x.

10. Stenhouse SA, Plernsub S, Yanola J, Lumjuan N, Dantrakool A, Choochote W, et al. Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand. Parasites & Vectors. 2013;6:1-10. doi:10.1186/1756-3305-6-253.

11. Bowman NM, Akialis K, Cave G, Barrera R, Apperson CS, Meshnick SR. Pyrethroid insecticides maintain repellent effect on knock-down resistant populations of Aedes aegypti mosquitoes. PLoS One. 2018;13(5):1-14. doi:10.1371/journal.pone.0196410.

12. Pichler V, Mancini E, Micocci M, Calzetta M, Arnoldi D, Rizzoli A, et al. A novel allele specific polymerase chain reaction (AS-PCR) assay to detect the V1016G knockdown resistance mutation confirms its widespread presence in Aedes albopictus populations from Italy. Insects. 2021;12(1):1-12. doi:10.3390/insects12010079.

13. Geneaid. Genomic DNA Mini Kit (Blood/Cultured Cell). 2020:1-2. Available from: https://www.geneaid.com/data/download/attached/16027431248%0A08219869.pdf%0A.

14. Galuszynski NC, Potts AJ. Application of high resolution melt analysis (HRM) for screening haplotype variation in a non-model plant genus: Cyclopia (honeybush). PeerJ. 2020;8:1-26. doi:10.7717/peerj.9187.

15. Widiastuti D, Agustiningsih. V1016G and F1534C mutation in the VGSC gene of cypermethrin resistant Aedes aegypti from Central Java. Adv Sci Lett. 2017;23(4):3309-12. doi:10.1166/asl.2017.9178.

16. Heydari N, Alikhani MY, Tahmasebi H, Asghari B, Arabestani MR. Design of melting curve analysis (MCA) by real-time polymerase chain reaction assay for rapid distinction of staphylococci and antibiotic resistance. Arch Clin Infect Dis. 2019;14(2):1-7. doi:10.5812/archcid.81604.

17. Ahmed FE, Gouda MM, Hussein LA, Ahmed NC, Vos PW, Mohammad MA. Role of melt curve analysis in interpretation of nutrigenomics’ microRNA expression data. Cancer Genomics Proteomics. 2017;14(6):469-81. doi:10.21873/cgp.20057.

18. Rajatileka S, Black 4th WC, Saavedra-Rodriguez K, Trongtokit Y, Apiwathnasorn C, McCall PJ, et al. Development and application of a simple colorimetric assay reveals widespread distribution of sodium channel mutations in Thai populations of Aedes aegypti. Acta Trop. 2008;108(1):54-7. doi:10.1016/j.actatropica.2008.08.004.

19. Sayono S, Hidayati APN, Fahri S, Sumanto D, Dharmana E, Hadisaputro S, et al. Distribution of voltage-gated sodium channel (Nav) alleles among the Aedes aegypti populations in Central Java Province and its association with resistance to pyrethroid insecticides. PLoS One. 2016;11(3):1-12. doi:10.1371/journal.pone.0150577.

20. Kawada H, Higa Y, Komagata O, Kasai S, Tomita T, Yen NT, et al. Widespread distribution of a newly found point mutation in voltage-gated sodium channel in pyrethroid-resistant Aedes aegypti populations in Vietnam. PLoS Negl Trop Dis. 2009;3(10):1-7. doi:10.1371/journal.pntd.0000527.

21. Chang C, Shen WK, Wang TT, Lin YH, Hsu EL, Dai SM. A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. Insect Biochem Mol Biol. 2009;39(4):272-8. doi:10.1016/j.ibmb.2009.01.001.
Published
2021-12-14
How to Cite
1.
Widiastuti D, Agustiningsih A, Ratna Sari I, Ramadhani T. Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel. blb [Internet]. 14Dec.2021 [cited 19May2022];17(2):153-60. Available from: https://ejournal2.litbang.kemkes.go.id/index.php/blb/article/view/5283
Section
Articles