Detecting wMel Wolbachia in field-collected Aedes aegypti mosquitoes using loop-mediated isothermal amplification (LAMP)



The World Mosquito Program uses Wolbachia pipientis for the biocontrol of arboviruses transmitted by Aedes aegypti mosquitoes. Diagnostic testing for Wolbachia in laboratory colonies and in field-caught mosquito populations has typically employed PCR. New, simpler methods to diagnose Wolbachia infection in mosquitoes are required for large-scale operational use.


Field-collected Ae. aegypti mosquitoes from North Queensland were tested using primers designed to detect the Wolbachia wsp gene, specific to the strain wMel. The results were analysed by colour change in the reaction mix. Furthermore, to confirm the efficiency of the LAMP assay, the results were compared to the gold-standard qPCR test.


A novel loop-mediated isothermal amplification (LAMP) colorimetric test for the wMel strain of Wolbachia was designed, developed and validated for use in a high-throughput setting. Against the standard qPCR test, the analytical sensitivity, specificity and diagnostic metrics were: sensitivity (99.6%), specificity (92.2%), positive predictive value (97.08%) and negative predictive value (99.30%).


We describe an alternative, novel and high-throughput method for diagnosing wMel Wolbachia infections in mosquitoes. This assay should support Wolbachia surveillance in both laboratory and field populations of Ae. aegypti.


Arboviral diseases transmitted by the mosquito Aedes aegypti such as dengue, chikungunya and Zika constitute a significant burden to human health and economic development worldwide [1, 2]. This is reflected in the nomination by the World Health Organisation of dengue as one of the top ten global health threats in 2019. There is an urgent need for novel and efficient strategies to control these diseases [3].

The World Mosquito Program (WMP,, formerly known as the Eliminate Dengue Program) has developed a novel arboviral disease biocontrol strategy utilising the endosymbiotic bacterium Wolbachia pipientis. This maternally transmitted bacterium [4] is found in 40–60% of insect species worldwide [5,6,7]. WMP reported the successful introduction of the wMel strain of Wolbachia into Ae. aegypti in northern Australia since 2011 [8]. Subsequently, numerous studies demonstrated that the presence of wMel reduces dengue virus [9], Zika [10], chikungunya virus [11] yellow fever [11] and Mayaro virus [12] infection and replication and, in turn the virus transmission potential of the mosquito [13, 14]. wMel has been established in field populations of Ae. aegypti in five countries and it has been expanded to more than ten worldwide [15]. The mitigation of local dengue outbreaks in northern Australia [16] following the establishment of wMel is consistent with the laboratory and modelling expectations of this intervention [17].

A duplex TaqManTM qPCR assay has been considered the gold-standard reference method for diagnosing Wolbachia infection in mosquitoes [5, 18]. The advantages of qPCR are clear: it is able to detect multiple genes of interest, produce quantitative or qualitative data [19] and is scalable. However, these advantages are significantly offset by high initial start-up costs and on-going maintenance of equipment, and the complexity of interpreting threshold values and amplification curves requires extensive training [20]. This is exacerbated in low-resource settings where the sourcing of laboratory equipment can be challenging, and technical expertise is not readily available. Given the rapid expansion of the World Mosquito Program [21, 22], there is a need to develop diagnostic tools suitable for resource limited settings.

Loop-mediated isothermal amplification (LAMP) [23, 24] is a technology potentially suitable for Wolbachia diagnostics in resource-limited laboratories [25, 26]. LAMP has been adapted as a nucleic acid test with multiple direct and indirect methods to detect several pathogens [27,28,29,30]. Hence the purpose of this study was to develop and validate the diagnostic accuracy of a colorimetric LAMP assay for the wMel strain of Wolbachia.


Mosquito samples

Field-collected Ae. aegypti mosquitoes (n = 3585) were collected from BG-Sentinel traps during and after Wolbachia establishment in Cairns, Townsville and Innisfail, Australia, over an eight-month period from June 2017. The DNA from each mosquito was individually crudely extracted in the laboratory as previously described [31] before being tested by both TaqManTM qPCR [32] and LAMP assays.

wMel LAMP reactions

LAMP primers (Integrated DNA Technologies, Singapore, Singapore) were designed to detect the wsp gene from wMel and wMelPop-CLA strains using the software LAMP Designer 1.02 (PREMIER Biosoft International, Horsham, UK). Individual reaction consisted of 2X WarmStart® Colorimetric LAMP Master Mix (New England BioLabs, Ipswich, USA; Cat# M1800S), primers according to the manufacturer recommendation (Table 1), and 1 μl of target DNA in a total reaction volume of 17 μl. Reactions for individual samples were performed in 96-well PCR plates (LabAdvantage, Tingalpa, Australia; 96-well PCR plates, full skirt, clear). Plates were incubated in a thermocycler (BioRad C1000) at 65 °C for 30 min then held at 12 °C until scoring. Within one hour after incubation, colour changes of individual samples were recorded where pink indicates negative, yellow as positive and orange as equivocal (see Additional file 1: Figure S1). Results were interpreted by the naked eye directly from the reaction plates and also captured with a smartphone for data storage.

Table 1 LAMP primers targeting wMel and wMelPop Wolbachia strains

wMel LAMP assay performance

Target specificity

To assess the specificity of the LAMP assay for wMel, multiple Wolbachia trans-infected Ae. aegypti lines (wMel, wMelPop-CLA, wRi, wPip and wAlbB) were tested, in addition to two tetracycline-treated lines (wMel.tet and wAlbB.tet). For each mosquito line, three mosquitos were tested and three technical replicates were performed, totalling nine reactions per line. Mosquitoes were reared and maintained as described [32], DNA was crudely extracted as previously described [31] and tested in the wMel LAMP assay.

Diagnostic performance comparison

Field-collected samples were tested using both the wMel LAMP and the reference TaqManTM qPCR assays [32]. The qPCR assay was modified by the use of an LC-640-tagged wsp gene probe (fluorescence bandwidth 618–660 nm) to provide improved separation from the FAM-tagged housekeeping gene Rps17 (fluorescence bandwidth 465–510 nm). Both the qPCR and the LAMP results were analysed in blinded fashion, i.e. the interpretation of each assay was performed blind to the outcome of the other assay. The LAMP assay diagnostic specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) calculations were performed with any equivocal samples (orange colour) being excluded from the calculations. All statistical analysis and calculations were performed using Medcalc (


wMel LAMP assay performance

Target specificity

The wMel LAMP assay consistently detected only the wsp target sequence from the wMel strain in Ae. aegypti, and did not amplify the other six Wolbachia strains tested (Additional file 2: Figure S2).

Diagnostic performance on field-caught mosquitoes

A total of 3585 individual field-collected adult mosquitoes, sampled between June 2017 to February 2018 from Cairns, Townsville and Innisfail, were tested by both TaqManTM qPCR and LAMP and the comparison between the results from each assay is shown in Table 2. Amongst the field-caught mosquitoes, 24 (0.7%) were found to be non-Ae. aegypti due to non-amplification of Ae. aegypti housekeeping Rps17 gene in duplex TaqManTM qPCR assay, and hence excluded from the analysis.

Table 2 wMel LAMP positivity and negativity compared to qPCR

Relative to the qPCR reference method, LAMP false positives were more likely (n = 70) to occur than false negatives (n = 8). Equivocal LAMP results were more likely to result from qPCR-negative samples than qPCR-positive samples. LAMP increased the estimation of wMel positivity by 2%, with an additional 1% of total samples producing equivocal results. The sensitivity of LAMP assay was close to 100% (Table 3) and this parameter was confirmed when performed in serially diluted field samples up to 1:1000 (data not shown). Also, wMel LAMP diagnostic had high positive and negative predictive values in relation to the wMel TaqManTM qPCR (Table 3).

Table 3 LAMP diagnostic parameters of LAMP-qPCR parallel testing


TaqManTM qPCR has been a mainstay for diagnosing Wolbachia infection in mosquitoes despite utilising expensive reagents and sophisticated equipment that require specialised training and maintenance [20]. Previous work has shown that there is potential to use LAMP for detecting Wolbachia in Ae. aegypti, either for any strain, targeting the 16S rRNA gene [25], or specifically targeting the wsp gene of the strains wAlbB and wPip [26]. Here, we have taken this framework and built on it by utilising a pH indicator that possesses the same characteristics but gives a greater resolution to differentiate between positive and negative results. The colorimetric LAMP assay in this study is an attractive candidate to replace qPCR because it does not require sophisticated equipment, is qualitative in nature, can easily be analysed by visual inspection and can be more cost-effective [33,34,35]. In addition, LAMP has been shown to be a reliable and robust assay across a range of DNA matrices [36] making it ideal for field-caught mosquito homogenates that can be highly variable. A small number of results were scored as false negatives. These could be explained by pipetting errors, or the presence of inhibitors of DNA amplification. Inhibitors such as EDTA, or human blood in blood-fed female mosquitoes, could block enzyme activity [37]. The frequency of false negatives was very low (0.22%), and does not affect the robustness of our assay.

When considering the implementation of colorimetric LAMP as the primary diagnostic method for monitoring the establishment of wMel, certain trade-offs should be recognised. First, compared to qPCR there may be an increased likelihood of contamination due to the high amplification efficiency of LAMP [23, 38]. Secondly, as the colorimetric LAMP assay is a single target nucleic acid test, it relies on entomologists to accurately identify Ae. aegypti mosquitoes from other species and insects that might be collected from the field. Thirdly, despite its robustness, the LAMP assay can produce equivocal results occasionally, presenting as wells with varying hues of the colour orange. However, equivocal results were rare (typically 1% of the samples) and did not significantly impact on the predictive ability of the assay. In general, this rate of equivocal findings should not adversely affect the chronological and geographical picture of Wolbachia establishment. Finally, the wMel LAMP assay is scored visually which may be subject to interpretation bias. To avoid possible visual biases, a smartphone application has been developed to conveniently and reliably score positivity and negativity, and this can promote consistency across multiple and international settings.


In conclusion, the wMel LAMP assay described here was sensitive, specific and suitable for high throughput application. With these results, we believe the assay is an appropriate tool to monitor the progress of wMel Wolbachia establishment in field Ae. aegypti populations worldwide in order to protect local communities from mosquito-borne diseases.

Availability of data and materials

All relevant data generated or analysed during this study are included in this published article and its additional files.


  1. 1.

    Hotez PJ. Forgotten people, forgotten diseases: The neglected tropical diseases and their impact on global health and development. Washington, DC: American Society for Microbiology Press; 2008.

    Google Scholar 

  2. 2.

    Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496:504–7.

    CAS  Article  Google Scholar 

  3. 3.

    Kyle JL, Harris E. Global spread and persistence of dengue. Annu Rev Microbiol. 2008;62:71–92.

    CAS  Article  Google Scholar 

  4. 4.

    Veneti Z, Clark ME, Zabalou S, Karr TL, Savakis C, Bourtzis K. Cytoplasmic incompatibility and sperm cyst infection in different Drosophila-Wolbachia associations. Genetics. 2003;164:545–52.

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    de Oliveira CD, Goncalves DS, Baton LA, Shimabukuro PH, Carvalho FD, Moreira LA. Broader prevalence of Wolbachia in insects including potential human disease vectors. Bull Entomol Res. 2015;105:305–15.

    Article  Google Scholar 

  6. 6.

    Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH. How many species are infected with Wolbachia? A statistical analysis of current data. FEMS Microbiol Lett. 2008;281:215–20.

    CAS  Article  Google Scholar 

  7. 7.

    Zug R, Hammerstein P. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One. 2012;7:e3854.

    Article  Google Scholar 

  8. 8.

    Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH, Muzzi F, et al. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature. 2011;476:454–7.

    CAS  Article  Google Scholar 

  9. 9.

    Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, et al. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, chikungunya, and Plasmodium. Cell. 2009;139:1268–78.

    Article  Google Scholar 

  10. 10.

    Dutra HL, Rocha MN, Dias FB, Mansur SB, Caragata EP, Moreira LA. Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes. Cell Host Microbe. 2016;19:771–4.

    CAS  Article  Google Scholar 

  11. 11.

    Aliota MT, Walker EC, Uribe Yepes A, Velez ID, Christensen BM, Osorio JE. The wMel strain of Wolbachia reduces transmission of chikungunya virus in Aedes aegypti. PLoS Negl Trop Dis. 2016;10:e0004677.

    Article  Google Scholar 

  12. 12.

    Pereira TN, Rocha MN, Sucupira PHF, Carvalho FD, Moreira LA. Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus. Sci Rep. 2018;8:6889.

    Article  Google Scholar 

  13. 13.

    Ye YH, Carrasco AM, Frentiu FD, Chenoweth SF, Beebe NW, van den Hurk AF, et al. Wolbachia reduces the transmission potential of dengue-infected Aedes aegypti. PLoS Negl Trop Dis. 2015;9:e0003894.

    Article  Google Scholar 

  14. 14.

    Carrington LB, Tran BCN, Le NTH, Luong TTH, Nguyen TT, Nguyen PT, et al. Field- and clinically derived estimates of Wolbachia-mediated blocking of dengue virus transmission potential in Aedes aegypti mosquitoes. Proc Natl Acad Sci USA. 2018;115:361–6.

    CAS  Article  Google Scholar 

  15. 15.

    OʼNeill SL. The use of Wolbachia by the World Mosquito Program to interrupt transmission of Aedes aegypti transmitted viruses. Adv Exp Med Biol. 2018;1062:355–60.

    Article  Google Scholar 

  16. 16.

    Ritchie SA. Wolbachia and the near cessation of dengue outbreaks in Northern Australia despite continued dengue importations via travellers. J Travel Med. 2018;251:1–3.

    Google Scholar 

  17. 17.

    Ferguson NM, Kien DT, Clapham H, Aguas R, Trung VT, Chau TN, et al. Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti. Sci Transl Med. 2015;7:279ra37.

    Article  Google Scholar 

  18. 18.

    Joubert DA, O’Neill SL. Comparison of stable and transient Wolbachia infection models in Aedes aegypti to block dengue and West Nile viruses. PLoS Negl Trop Dis. 2017;11:e0005275.

    Article  Google Scholar 

  19. 19.

    Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 2009;67:6–20.

    CAS  Article  Google Scholar 

  20. 20.

    Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15:155–66.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Flores HA, OʼNeill SL. Controlling vector-borne diseases by releasing modified mosquitoes. Nat Rev Microbiol. 2018;16:508–18.

    CAS  Article  Google Scholar 

  22. 22.

    OʼNeill S, Ryan P, Turley A, Wilson G, Retzki K, Iturbe-Ormaetxe I, et al. Scaled deployment of Wolbachia to protect the community from Aedes transmitted arboviruses. Gates Open Res. 2018;2:36.

    Article  Google Scholar 

  23. 23.

    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63.

    CAS  Article  Google Scholar 

  24. 24.

    Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009;15:62–9.

    CAS  Article  Google Scholar 

  25. 25.

    Goncalves Dda S, Cassimiro AP, de Oliveira CD, Rodrigues NB, Moreira LA. Wolbachia detection in insects through LAMP: loop mediated isothermal amplification. Parasit Vectors. 2014;7:228.

    Article  Google Scholar 

  26. 26.

    Bhadra S, Riedel TE, Saldana MA, Hegde S, Pederson N, Hughes GL, et al. Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. PLoS Negl Trop Dis. 2018;12:e0006671.

    Article  Google Scholar 

  27. 27.

    Calvert AE, Biggerstaff BJ, Tanner NA, Lauterbach M, Lanciotti RS. Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP). PLoS One. 2017;12:e0185340.

    Article  Google Scholar 

  28. 28.

    Khan MG, Bhaskar KR, Salam MA, Akther T, Pluschke G, Mondal D. Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients. Parasit Vectors. 2012;5:280.

    CAS  Article  Google Scholar 

  29. 29.

    Neeraja M, Lakshmi V, Lavanya V, Priyanka EN, Parida MM, Dash PK, et al. Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India. J Virol Methods. 2015;211:22–31.

    CAS  Article  Google Scholar 

  30. 30.

    Quoc NB, Phuong NDN, Chau NNB, Linh DTP. Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood. Heliyon. 2018;4:e00561.

    Article  Google Scholar 

  31. 31.

    Yeap HL, Axford JK, Popovici J, Endersby NM, Iturbe-Ormaetxe I, Ritchie SA, et al. Assessing quality of life-shortening Wolbachia-infected Aedes aegypti mosquitoes in the field based on capture rates and morphometric assessments. Parasit Vectors. 2014;7:58.

    Article  Google Scholar 

  32. 32.

    Frentiu FD, Zakir T, Walker T, Popovici J, Pyke AT, van den Hurk A, et al. Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia. PLoS Negl Trop Dis. 2014;8:e2688.

    Article  Google Scholar 

  33. 33.

    Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. 2009;46:167–72.

    CAS  Article  Google Scholar 

  34. 34.

    Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16:223–9.

    CAS  Article  Google Scholar 

  35. 35.

    Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP): recent progress in research and development. J Inf Chem. 2013;19:404–11.

    CAS  Google Scholar 

  36. 36.

    Francois P, Tangomo M, Hibbs J, Bonetti EJ, Boehme CC, Notomi T, et al. Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications. FEMS Immunol Med Microbiol. 2011;62:41–8.

    CAS  Article  Google Scholar 

  37. 37.

    Oscorbin IP, Belousova EA, Boyarskikh UA, Zakabunin AI, Khrapov EA, Filipenko ML. Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance. Nucleic Acids Res. 2017;45:9595–610.

    CAS  Article  Google Scholar 

  38. 38.

    Parida M, Sannarangaiah S, Dash PK, Rao PV, Morita K. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol. 2008;18:407–21.

    CAS  Article  Google Scholar 

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We thank the World Mosquito Program Oceania field deployment team for collecting mosquito samples; NEB for assistance; Luciano Andrade Moreira for providing outstanding technical assistance; Jesse Chapotet for helping to process samples; Heather A. Flores and David C. Hack for critical reading, and Stephanie Tanamas for statistical advice.


This work was supported by Wellcome trust strategic award.

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DSG, DJH, NB and PK participated to the study design, performed experiments, contributed to obtaining the results and drafted the manuscript. YD contributed to the study design and drafted the manuscript. IIO, CPM and SLO participated on the study design. CPM and SLO coordinated the work and critically reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Daniela da Silva Gonçalves.

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Additional files

Additional file 1: Figure S1.

Example of colorimetric LAMP result interpretation. Results are scored based on colour change. Samples (1) and (2) in yellow are positive for wMel Wolbachia; (3) and (4) in pink are negative; and (5) and (6) in orange are considered equivocal.

Additional file 2: Figure S1.

Specificity of the wMel LAMP assay. LAMP reactions were performed using a number of Ae. aegypti lines transinfected with different Wolbachia strains per column, as follows: (1) wMel-infected, field-collected; (2) wMel, purified gDNA; (3) wAlbB; (4) Ae. aegypti tetracycline treated (without wAlbB); (5) wMelPop-CLA; (6) wPip; (7) wRi; (8) wMelCS; (9) Ae. aegypti tetracycline treated (without wMel); (10) wild type uninfected Ae. aegypti from Townsville, Australia; (11) water; and (12) extraction buffer negative control. Eight technical replicates were run for controls.

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Gonçalves, D.S., Hooker, D.J., Dong, Y. et al. Detecting wMel Wolbachia in field-collected Aedes aegypti mosquitoes using loop-mediated isothermal amplification (LAMP). Parasites Vectors 12, 404 (2019).

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  • wMel
  • Wolbachia
  • LAMP
  • Diagnostics