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. 2021 Sep 23;16(9):e0257781.
doi: 10.1371/journal.pone.0257781. eCollection 2021.

A diagnostic primer pair to distinguish between wMel and wAlbB Wolbachia infections

Meng-Jia Lau  1 , Ary A Hoffmann  1 , Nancy M Endersby-Harshman  1
Affiliations

A diagnostic primer pair to distinguish between wMel and wAlbB Wolbachia infections

Meng-Jia Lau et al. PLoS One. .

Abstract

Detection of the Wolbachia endosymbiont in Aedes aegypti mosquitoes through real-time polymerase chain reaction assays is widely used during and after Wolbachia releases in dengue reduction trials involving the wMel and wAlbB strains. Although several different primer pairs have been applied in current successful Wolbachia releases, they cannot be used in a single assay to distinguish between these strains. Here, we developed a new diagnostic primer pair, wMwA, which can detect the wMel or wAlbB infection in the same assay. We also tested current Wolbachia primers and show that there is variation in their performance when they are used to assess the relative density of Wolbachia. The new wMwA primers provide an accurate and efficient estimate of the presence and density of both Wolbachia infections, with practical implications for Wolbachia estimates in field collected Ae. aegypti where Wolbachia releases have taken place.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Development of primers to detect Wolbachia wMel and wAlbB infection in Aedes aegypti.
(a) The new primer pair wMwA aligns to a region in gene WD_RS06155 of wMel, and also aligns to its analogue in the wAlbB genome which has two base-pair mismatches at the 3’- end; (b) the wMwA primers showed distinct Tm peaks for Wolbachia wMel and wAlbB. (c) the wMwA primers showed distinct Tm values for Wolbachia wMel (82.6 ± 0.03°C) and wAlbB (80.4 ± 0.02°C), the x axis represents the quantification cycle (Cq) and the y axis represents the amplicon melting temperature; (d) the wMwA primers showed two Tm peaks when mixing DNA templates of wMel and wAlbB-infected Ae. aegypti.
Fig 2
Fig 2. Primer efficiency for detection of Wolbachia strains and estimation of density.
DNA was extracted in 250 μL 5% Chelex® 100 Resin and then diluted ten times before making a three-fold dilution series. The primer names are defined in Table 2.
Fig 3
Fig 3. Variation in the shape of the PCR amplification curves.
The curves from left to right represent amplification curves of 1/10, 1/30, 1/90, 1/270 and 1/810 DNA dilution from initial extraction in 250 μL 5% Chelex® 100 Resin. The primers are defined in Table 2.
Fig 4
Fig 4. Variation in Cq values when using different Wolbachia primers for the same samples.
Correlation of Cq values between (a) wA and wsp primers in Wolbachia wAlbB screening; (b) wMwA and wsp primers in Wolbachia wAlbB screening; (c) mos and aeg primers in Aedes aegypti screening; (d) wM and wsp primers in Wolbachia wMel screening; (e) wMwA and wsp primers in Wolbachia wMel screening; (f) w1 and wsp primers in Wolbachia wMel screening.
See this image and copyright information in PMC

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