Echeverry Diego F, Deason Nicholas A, Makuru Victoria, Davidson Jenna, Xiao Honglin, Niedbalski Julie, Yu Xiaoyu, Stevenson Jennifer C, Bugoro Hugo, Aparaimo Allan, Reuben Hedrick, Cooper Robert, Burkot Thomas R, Russell Tanya L, Collins Frank H, Lobo Neil F
Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA.
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Malar J. 2017 May 31;16(1):230. doi: 10.1186/s12936-017-1881-1.
Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming.
In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330-0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax.
The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands.
This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination.
需要具备更高实用性、灵敏度、特异性以及高通量的检测感染疟疾蚊子的分子工具。一种用于检测感染疟原虫属蚊子的常见PCR技术是基于18s - rRNA基因的巢式PCR检测法。然而，该技术存在一些技术局限性，操作繁琐且耗时。
在本研究中，将基于疟原虫细胞色素氧化酶I（COX - I）基因的PCR与使用间日疟原虫、恶性疟原虫和诺氏疟原虫DNA系列稀释液（330 - 0.0012 pg）以及来自48只肯尼亚阳性和阴性蚊子（先前通过ELISA和PCR检测）的DNA进行的18s - rRNA巢式PCR相比较。然后，使用快速COX - I PCR检测法对从2122只野外采集的蚊子（来自所罗门群岛）的头部和胸部提取的DNA单独进行疟原虫属DNA检测。
快速COX - I PCR检测法运行耗时1小时，在单次PCR中始终能检测到低至0.043 pg的寄生虫DNA（相当于两个寄生虫），而18s - rRNA巢式PCR分析需要4小时完成，一致检测阈值为1.5 pg DNA。当应用于48个已知疟原虫属感染状态的肯尼亚对照样本时，两种检测法结果一致。快速COX - I PCR从所罗门群岛鉴定出23/2122只感染疟原虫的蚊子。
这种适用于单次PCR反应的新型COX - I PCR是一种更快、更简单、更便宜、更灵敏的技术，适用于高通量分析蚊子中的疟原虫DNA检测，并且与18s - rRNA巢式PCR相当。快速COX - I PCR所显示的更高灵敏度将提高蚊子感染率测定的准确性。
