Molecular Ecology and Health Laboratory, Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama, Japan.
Entomology Laboratory, Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Front Cell Infect Microbiol. 2023 Dec 14;13:1252656. doi: 10.3389/fcimb.2023.1252656. eCollection 2023.
, an endosymbiotic bacterium, is globally used to control arboviruses because of its ability to block arboviral replication and manipulate the reproduction of host, . Polymerase chain reaction (PCR)-based detection has been recently reported from natural populations. However, due to the technical limitations of PCR, such as primer incompatibility, PCR-based assays are not sufficiently reliable or accurate. In this study, we examined double digestion restriction site-associated DNA sequencing (ddRAD-Seq) efficiency and limitations in detection and quantification in field-collected natural populations in Metro Manila, the Philippines, compared with PCR-based assays.
A total of 217 individuals were collected from Metropolitan Manila, Philippines. We separated it into 14 populations consisting of 7 female and male populations. We constructed a library for pool ddRAD-Seq per population and also screened for by PCR assays using and rRNA. density per population were measured using as the housekeeping gene.
From 146,239,637 sequence reads obtained, 26,299 and 43,778 reads were mapped across the entire genome (with the AlbA and AlbB strains, respectively), suggesting that ddRAD-Seq complements PCR assays and supports more reliable detection from a genome-wide perspective. The number of reads mapped to the genome per population positively correlated with the number of -infected individuals per population based on PCR assays and the relative density of in the populations based on qPCR, suggesting ddRAD-Seq-based semi-quantification of by ddRAD-Seq. Male exhibited more reads mapped to the genome than females, suggesting higher prevalence rates in their case. We detected 150 single nucleotide polymorphism loci across the genome, allowing for more accurate the detection of four strains: PipRi, and Mel.
Taken together, our results demonstrate the feasibility of ddRAD-Seq-based detection from field-collected mosquitoes.
，一种内共生菌，因其能够阻断虫媒病毒的复制并操纵 宿主的繁殖而在全球范围内被用于控制虫媒病毒。最近已经从自然种群中报道了基于聚合酶链反应（PCR）的 检测。然而，由于 PCR 的技术限制，例如引物不兼容，基于 PCR 的检测并不足够可靠或准确。在这项研究中，我们检查了双消化限制性位点相关 DNA 测序（ddRAD-Seq）在检测和定量菲律宾马尼拉大都市地区野外采集的 自然种群中的效率和局限性，与基于 PCR 的检测相比。
从菲律宾马尼拉大都会共收集了 217 只 个体。我们将其分为 14 个种群，每个种群由 7 个雌性和雄性种群组成。我们为每个种群的池 ddRAD-Seq 构建了一个文库，并使用 和 rRNA 进行了 PCR 检测。使用 作为管家基因测量每个种群的 密度。
从获得的 146,239,637 个序列读段中，有 26,299 和 43,778 个读段分别映射到整个 基因组（分别为 AlbA 和 AlbB 菌株），这表明 ddRAD-Seq 补充了 PCR 检测，并从全基因组角度支持更可靠的 检测。基于 PCR 检测，每个种群感染个体的数量与每个种群的 基因组读段数量呈正相关，基于 qPCR 检测 种群的 相对密度也呈正相关，表明 ddRAD-Seq 可以基于半定量检测 。雄性 比雌性映射到 基因组的读段更多，这表明它们的 感染率更高。我们在 基因组上检测到 150 个单核苷酸多态性位点，允许更准确地检测四种菌株：PipRi、和 Mel。
总之，我们的结果表明了从野外采集的 蚊子中进行基于 ddRAD-Seq 的 检测的可行性。
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