Author:
- ABSTRACT.
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Species-specific identification of major mosquito vectors and confirmation through Sanger sequencing.
- Pan-specificity of the primer sets.
- One-step multiplex PCR assay for the identification of major vector mosquitoes.
- Quantity assessment and detection of limit for species-specific ITS2 gene amplification.
- DISCUSSION
- ACKNOWLEDGMENTS
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
World Health Organization, 2020. Vector-borne Diseases. Available at: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. Accessed March 28, 2024.
-
2.
Center For Science Education, 2024. Climate Change and Vector-borne Disease. Available at: https://scied.ucar.edu/learning-zone/climate-change-impacts/vector-borne-disease. Accessed March 28, 2024.
-
3.
World Health Organization, 2021. World Malaria Report 2021. Geneva, Switzerland: WHO.
-
4.
World Health Organization, 2022. Virtual Meeting Report 2022. Virtual Meeting of Regional Technical Advisory Group for Dengue and Other Arbovirus Diseases, Regional Office for South-East Asia. New Delhi, India, 4–6 October 2021. Geneva, Switzerland: WHO.
-
5.
Messina JP et al., 2019. The current and future global distribution and population at risk of dengue. Nat Microbiol 4: 1508–1515.
-
6.
Barredo E, DeGennaro M, 2020. Not just from blood: Mosquito nutrient acquisition from nectar sources. Trends Parasitol 36: 473–484.
-
7.
Nyasembe VO, Tchouassi DP, Muturi MN, Pirk CW, Sole CL, Torto B, 2021. Plant nutrient quality impacts survival and reproductive fitness of the dengue vector Aedes aegypti. Parasit Vectors 14: 4.
-
8.
Nikbakhtzadeh MR, Terbot JW, Foster WA, 2016. Survival value and sugar access of four East African plant species attractive to a laboratory strain of sympatric Anopheles gambiae (Diptera: Culicidae). J Med Entomol 53: 1105–1111.
-
9.
World Health Organization, 2022. Sixteenth Meeting of the WHO Vector Control Advisory Group. Geneva, Switzerland: WHO.
-
10.
World Health Organization, Regional Office for South-East Asia, 2020. Pictorial Identification Key of Important Disease Vectors in the WHO South-East Asia Region. New Dehli, India: WHO, Regional Office for South-East Asia.
-
11.
Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, Velayudhan R, Corbel V, 2018. Integrated Aedes management for the control of Aedes-borne diseases. PLoS Negl Trop Dis 12: e0006845.
-
12.
Wilson AL, Courtenay O, Kelly-Hope LA, Scott TW, Takken W, Torr SJ, Lindsay SW, 2020. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis 14: e0007831.
-
13.
World Health Organization, 2017. How to Design Vector Control Efficacy Trials: Guidance on Phase III Vector Control Field Trial Design. Geneva, Switzerland: WHO.
-
14.
Hill CA, Kafatos FC, Stansfield SK, Collins FH, 2005. Arthropod-borne diseases: Vector control in the genomics era. Nat Rev Microbiol 3: 262–268.
-
15.
Takken W, Knols BG, 2009. Malaria vector control: Current and future strategies. Trends Parasitol 25: 101–104.
-
16.
Tyagi BK, Munirathinam A, Venkatesh A, 2015. A catalogue of Indian mosquitoes. Int J Mosq Res 2: 50–97.
-
17.
Garros C, Harbach RE, Manguin S, 2005. Morphological assessment and molecular phylogenetics of the Funestus and Minimus Groups of Anopheles (Cellia). J Med Entomol 42: 522–536.
-
18.
Wilkerson RC, Reinert JF, Li C, 2004. Ribosomal DNA ITS2 sequences differentiate six species in the Anopheles crucians complex (Diptera: Culicidae). J Med Entomol 41: 392–401.
-
19.
Prakash A, Walton C, Bhattacharyya DR, O’Loughlin S, Mohapatra PK, Mahanta J, 2006. Molecular characterization and species identification of the Anopheles dirus and Anopheles minimus complexes in north-east India using r-DNA ITS-2. Acta Trop 100: 156–161.
-
20.
Prakash A, Walton C, Bhattacharyya DR, Mohapatra PK, Mahanta J, 2006. Characterization of ITS2 rDNA of Anopheles philippinensis and Anopheles nivipes (Diptera: Culicidae) from north-east India. Southeast Asian J Trop Med Public Health 37: 1134–1138.
-
21.
Das R, Vashisht K, Pandey KC, 2023. A novel multiplex qPCR assay for clinical diagnosis of non-human malaria parasites-Plasmodium knowlesi and Plasmodium cynomolgi. Front Vet Sci 10: 1127273.
-
22.
Das M, Das MK, Dutta P, 2016. Genetic characterization and molecular phylogeny of Aedes albopictus (Skuse) species from Sonitpur district of Assam, India based on COI and ITS1 genes. J Vector Borne Dis 53: 240–247.
-
23.
MartÃnez D, Hernández C, Muñoz M, Armesto Y, Cuervo A, RamÃrez JD, 2020. Identification of Aedes (Diptera: Culicidae) species and arboviruses circulating in Arauca, Eastern Colombia. Front Ecol Evol 8: 602190.
-
24.
Tahir HM, Mehwish Kanwal N, 2016. The sequence divergence in cytochrome C oxidase I gene of Culex quinquefasciatus mosquito and its comparison with four other Culex species. Mitochondrial Dna A Dna Mapp Seq Anal 27: 3054–3057.
-
25.
Singh OP, Kaur T, Sharma G, Kona MP, Mishra S, Kapoor N, Mallick PK, 2023. Molecular tools for early detection of invasive malaria vector Anopheles stephensi mosquitoes. Emerg Infect Dis 29: 36–44.
-
26.
Kumar G, Gupta SK, Rahi M, Sharma A, 2022. Challenges in understanding the bionomics of Indian malaria vectors. Am J Trop Med Hyg 107: 1005–1014.
-
27.
Sriwichai P, Karl S, Samung Y, Sumruayphol S, Kiattibutr K, Payakkapol A, Mueller I, Yan G, Cui L, Sattabongkot J, 2015. Evaluation of CDC light traps for mosquito surveillance in a malaria endemic area on the Thai-Myanmar border. Parasit Vectors 8: 636.
-
28.
Odetoyinbo JA, 1969. Preliminary investigation on the use of a light-trap for sampling malaria vectors in the Gambia. Bull World Health Organ 40: 547–560.
-
29.
Kittichai V, Pengsakul T, Chumchuen K, Samung Y, Sriwichai P, Phatthamolrat N, Tongloy T, Jaksukam K, Chuwongin S, Boonsang S, 2021. Deep learning approaches for challenging species and gender identification of mosquito vectors. Sci Rep 11: 4838.
-
30.
Hien DFDS et al., 2016. Plant-mediated effects on mosquito capacity to transmit human malaria. PLoS Pathog 12: e1005773.
-
31.
Lardeux F, Tejerina R, Aliaga C, Ursic-Bedoya R, Lowenberger C, Chavez T, 2008. Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia and its application to field epidemiological studies. Trans R Soc Trop Med Hyg 102: 485–492.
-
32.
Jangra S, Ghosh A, 2022. Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications. PLoS One 17: e0271312.
-
33.
Calderón-Cortés N, Quesada M, Cano-Camacho H, Zavala-Páramo G, 2010. A simple and rapid method for DNA isolation from xylophagous insects. Int J Mol Sci 11: 5056–5064.
-
34.
Bang WJ, Kim HC, Ryu J, Lee HS, Lee SY, Kim MS, Chong ST, Klein TA, Choi KS, 2021. Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris, and Lindesayi groups. Malar J 20: 287.
-
35.
Hung JH, Weng Z, 2016. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harb Protoc: 821–826.
-
36.
Dovichi NJ, 1997. DNA sequencing by capillary electrophoresis. Electrophoresis 18: 2393–2399.
-
37.
Meyer J, 2007. Gel Extraction Protocol (QIAquick Gel Extraction Kit Protocol). Available at: www.qiagen.com. Accessed November 12, 2024.
-
38.
Dzaki N, Ramli KN, Azlan A, Ishak IH, Azzam G, 2017. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti. Sci Rep 7: 43618.
-
39.
Karmakar S, Nath S, Sarkar B, Chakraborty S, Paul S, Karan M, Pal C, 2021. Insect vectors’ saliva and gut microbiota as a blessing in disguise: Probability versus possibility. Future Microbiol 16: 657–670.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1753 | 1753 | 652 |
| Full Text Views | 30 | 30 | 0 |
| PDF Downloads | 37 | 37 | 0 |
One-Step Multiplex Polymerase Chain Reaction Assay for the Detection of Major Disease-Transmitting Mosquito Vectors in India
Mintu Karan
Mintu Karan
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Sharmistha Paul
Sharmistha Paul
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Supriya Nath
Supriya Nath
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Bedanta Das
Bedanta Das
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Sanhita Ghosh
Sanhita Ghosh
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Suman Karmakar
Suman Karmakar
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Pritam Mandal
Pritam Mandal
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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Biplab Bhowmik
Biplab Bhowmik
Department of Zoology, Diamond Harbour Women´s University, Diamond Harbour, India;
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Piyoosh Kumar Singh
Piyoosh Kumar Singh
Field Unit, ICMR-National Institute of Malaria Research, Ranchi, India;
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Rajnikant Dixit
Rajnikant Dixit
ICMR-National Institute of Malaria Research, Dwarka, India
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Chiranjib Pal
chiranjibpal.zoology@wbsu.ac.in
Chiranjib Pal
Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India;
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ABSTRACT.
Mosquitoes are important vectors that transmit viral, protozoan, and helminthic diseases across the world. Climate change and unplanned urbanization are accelerating the spread of these diseases. Controlling vector-borne diseases can be performed most effectively through vector control. Inadequate knowledge of vector bionomics is an impediment and can lead to inappropriate vector control efforts. However, the conventional methods of vector identification are based on morphological differences, demand a significant amount of time and specific skills, and are often misleading. An efficient and affordable solution is needed to quickly and accurately identify pooled samples from vast geographical territories. To ensure the correct identification of distorted or pooled samples in India, a set of definitive steps is required, including the construction of unique primers and the standardization of a one-step assay based on the second internal transcribed spacer gene of the ribosomal DNA. We have successfully developed and confirmed a highly efficient one-step multiplex reverse transcriptase polymerase chain reaction assay for the accurate identification of major mosquito vectors, especially in the cases of both the adult and larval forms of Anopheles sp., Aedes sp., and Culex sp. Hence, the specificity, universality, and uniqueness of these primers could serve as a critical tool for the rapid one-step and one-reaction identification of mosquitoes to control mosquito-borne disease outbreaks and public health emergencies.
Author Notes
Financial support: This research was supported by
Authors’ contributions: M. Karan designed the PCR strategies, performed experiments, analyzed data, and wrote the first draft of the manuscript; S. Paul performed experiments and acquired the data; S. Nath, B. Das, S. Ghosh, S. Karmakar, and P. Mandal performed the experiments; S. Ghosh contributed to finalizing the manuscript; B. Bhowmik, P. K. Singh, and R. Dixit acquired the field samples; C. Pal designed the study, finalized the manuscript, and acquired funds; all authors reviewed the final version of the manuscript and approved it.
Current contact information: Mintu Karan, Sharmistha Paul, Supriya Nath, Bedanta Das, Sanhita Ghosh, Suman Karmakar, Pritam Mandal, and Chiranjib Pal, Cellular Immunology and Vector Molecular Biology Laboratory, Department of Zoology, West Bengal State University, Barasat, India, E-mails: mintukaran15@gmail.com, sharmistha.mistu92@gmail.com, suprion9@gmail.com, bedantadas47@gmail.com, mitti88.1@gmail.com, karmakarsuman77@gmail.com, pritam.mandal2295@gmail.com, and chiranjibpal.zoology@wbsu.ac.in or cpcu.immunology@gmail.com. Biplab Bhowmik, Department of Zoology, Diamond Harbour Women’s University, Diamond Harbour, India, E-mail: panchakotbb@gmail.com. Piyoosh Kumar Singh, Field Unit, ICMR-National Institute of Malaria Research, Ranchi, India, E-mail: drpksingh45@gmail.com. Rajnikant Dixit, ICMR-National Institute of Malaria Research, Dwarka, India, E-mails: dixitrk@mrcindia.org and dixit2k@yahoo.com.
- ABSTRACT.
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- Species-specific identification of major mosquito vectors and confirmation through Sanger sequencing.
- Pan-specificity of the primer sets.
- One-step multiplex PCR assay for the identification of major vector mosquitoes.
- Quantity assessment and detection of limit for species-specific ITS2 gene amplification.
- DISCUSSION
- ACKNOWLEDGMENTS
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
World Health Organization, 2020. Vector-borne Diseases. Available at: https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases. Accessed March 28, 2024.
-
2.
Center For Science Education, 2024. Climate Change and Vector-borne Disease. Available at: https://scied.ucar.edu/learning-zone/climate-change-impacts/vector-borne-disease. Accessed March 28, 2024.
-
3.
World Health Organization, 2021. World Malaria Report 2021. Geneva, Switzerland: WHO.
-
4.
World Health Organization, 2022. Virtual Meeting Report 2022. Virtual Meeting of Regional Technical Advisory Group for Dengue and Other Arbovirus Diseases, Regional Office for South-East Asia. New Delhi, India, 4–6 October 2021. Geneva, Switzerland: WHO.
-
5.
Messina JP et al., 2019. The current and future global distribution and population at risk of dengue. Nat Microbiol 4: 1508–1515.
-
6.
Barredo E, DeGennaro M, 2020. Not just from blood: Mosquito nutrient acquisition from nectar sources. Trends Parasitol 36: 473–484.
-
7.
Nyasembe VO, Tchouassi DP, Muturi MN, Pirk CW, Sole CL, Torto B, 2021. Plant nutrient quality impacts survival and reproductive fitness of the dengue vector Aedes aegypti. Parasit Vectors 14: 4.
-
8.
Nikbakhtzadeh MR, Terbot JW, Foster WA, 2016. Survival value and sugar access of four East African plant species attractive to a laboratory strain of sympatric Anopheles gambiae (Diptera: Culicidae). J Med Entomol 53: 1105–1111.
-
9.
World Health Organization, 2022. Sixteenth Meeting of the WHO Vector Control Advisory Group. Geneva, Switzerland: WHO.
-
10.
World Health Organization, Regional Office for South-East Asia, 2020. Pictorial Identification Key of Important Disease Vectors in the WHO South-East Asia Region. New Dehli, India: WHO, Regional Office for South-East Asia.
-
11.
Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, Velayudhan R, Corbel V, 2018. Integrated Aedes management for the control of Aedes-borne diseases. PLoS Negl Trop Dis 12: e0006845.
-
12.
Wilson AL, Courtenay O, Kelly-Hope LA, Scott TW, Takken W, Torr SJ, Lindsay SW, 2020. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis 14: e0007831.
-
13.
World Health Organization, 2017. How to Design Vector Control Efficacy Trials: Guidance on Phase III Vector Control Field Trial Design. Geneva, Switzerland: WHO.
-
14.
Hill CA, Kafatos FC, Stansfield SK, Collins FH, 2005. Arthropod-borne diseases: Vector control in the genomics era. Nat Rev Microbiol 3: 262–268.
-
15.
Takken W, Knols BG, 2009. Malaria vector control: Current and future strategies. Trends Parasitol 25: 101–104.
-
16.
Tyagi BK, Munirathinam A, Venkatesh A, 2015. A catalogue of Indian mosquitoes. Int J Mosq Res 2: 50–97.
-
17.
Garros C, Harbach RE, Manguin S, 2005. Morphological assessment and molecular phylogenetics of the Funestus and Minimus Groups of Anopheles (Cellia). J Med Entomol 42: 522–536.
-
18.
Wilkerson RC, Reinert JF, Li C, 2004. Ribosomal DNA ITS2 sequences differentiate six species in the Anopheles crucians complex (Diptera: Culicidae). J Med Entomol 41: 392–401.
-
19.
Prakash A, Walton C, Bhattacharyya DR, O’Loughlin S, Mohapatra PK, Mahanta J, 2006. Molecular characterization and species identification of the Anopheles dirus and Anopheles minimus complexes in north-east India using r-DNA ITS-2. Acta Trop 100: 156–161.
-
20.
Prakash A, Walton C, Bhattacharyya DR, Mohapatra PK, Mahanta J, 2006. Characterization of ITS2 rDNA of Anopheles philippinensis and Anopheles nivipes (Diptera: Culicidae) from north-east India. Southeast Asian J Trop Med Public Health 37: 1134–1138.
-
21.
Das R, Vashisht K, Pandey KC, 2023. A novel multiplex qPCR assay for clinical diagnosis of non-human malaria parasites-Plasmodium knowlesi and Plasmodium cynomolgi. Front Vet Sci 10: 1127273.
-
22.
Das M, Das MK, Dutta P, 2016. Genetic characterization and molecular phylogeny of Aedes albopictus (Skuse) species from Sonitpur district of Assam, India based on COI and ITS1 genes. J Vector Borne Dis 53: 240–247.
-
23.
MartÃnez D, Hernández C, Muñoz M, Armesto Y, Cuervo A, RamÃrez JD, 2020. Identification of Aedes (Diptera: Culicidae) species and arboviruses circulating in Arauca, Eastern Colombia. Front Ecol Evol 8: 602190.
-
24.
Tahir HM, Mehwish Kanwal N, 2016. The sequence divergence in cytochrome C oxidase I gene of Culex quinquefasciatus mosquito and its comparison with four other Culex species. Mitochondrial Dna A Dna Mapp Seq Anal 27: 3054–3057.
-
25.
Singh OP, Kaur T, Sharma G, Kona MP, Mishra S, Kapoor N, Mallick PK, 2023. Molecular tools for early detection of invasive malaria vector Anopheles stephensi mosquitoes. Emerg Infect Dis 29: 36–44.
-
26.
Kumar G, Gupta SK, Rahi M, Sharma A, 2022. Challenges in understanding the bionomics of Indian malaria vectors. Am J Trop Med Hyg 107: 1005–1014.
-
27.
Sriwichai P, Karl S, Samung Y, Sumruayphol S, Kiattibutr K, Payakkapol A, Mueller I, Yan G, Cui L, Sattabongkot J, 2015. Evaluation of CDC light traps for mosquito surveillance in a malaria endemic area on the Thai-Myanmar border. Parasit Vectors 8: 636.
-
28.
Odetoyinbo JA, 1969. Preliminary investigation on the use of a light-trap for sampling malaria vectors in the Gambia. Bull World Health Organ 40: 547–560.
-
29.
Kittichai V, Pengsakul T, Chumchuen K, Samung Y, Sriwichai P, Phatthamolrat N, Tongloy T, Jaksukam K, Chuwongin S, Boonsang S, 2021. Deep learning approaches for challenging species and gender identification of mosquito vectors. Sci Rep 11: 4838.
-
30.
Hien DFDS et al., 2016. Plant-mediated effects on mosquito capacity to transmit human malaria. PLoS Pathog 12: e1005773.
-
31.
Lardeux F, Tejerina R, Aliaga C, Ursic-Bedoya R, Lowenberger C, Chavez T, 2008. Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia and its application to field epidemiological studies. Trans R Soc Trop Med Hyg 102: 485–492.
-
32.
Jangra S, Ghosh A, 2022. Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications. PLoS One 17: e0271312.
-
33.
Calderón-Cortés N, Quesada M, Cano-Camacho H, Zavala-Páramo G, 2010. A simple and rapid method for DNA isolation from xylophagous insects. Int J Mol Sci 11: 5056–5064.
-
34.
Bang WJ, Kim HC, Ryu J, Lee HS, Lee SY, Kim MS, Chong ST, Klein TA, Choi KS, 2021. Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris, and Lindesayi groups. Malar J 20: 287.
-
35.
Hung JH, Weng Z, 2016. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harb Protoc: 821–826.
-
36.
Dovichi NJ, 1997. DNA sequencing by capillary electrophoresis. Electrophoresis 18: 2393–2399.
-
37.
Meyer J, 2007. Gel Extraction Protocol (QIAquick Gel Extraction Kit Protocol). Available at: www.qiagen.com. Accessed November 12, 2024.
-
38.
Dzaki N, Ramli KN, Azlan A, Ishak IH, Azzam G, 2017. Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti. Sci Rep 7: 43618.
-
39.
Karmakar S, Nath S, Sarkar B, Chakraborty S, Paul S, Karan M, Pal C, 2021. Insect vectors’ saliva and gut microbiota as a blessing in disguise: Probability versus possibility. Future Microbiol 16: 657–670.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1753 | 1753 | 652 |
| Full Text Views | 30 | 30 | 0 |
| PDF Downloads | 37 | 37 | 0 |