One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum
One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum
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DOI：
10.1371/journal.pone.0236369
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发表时间：
2020-07-23
期刊：
影响因子：
3.7
通讯作者：
Talundzic, Eldin
中科院分区：
文献类型：
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作者：
Jones, Sophie;Subramaniam, Gireesh;Talundzic, Eldin
富组氨酸蛋白2（HRP 2）检测快速诊断测试（RDT）在偏远地区迅速诊断疟疾方面发挥了重要作用。然而，pfhrp 2缺失的寄生虫的出现正在威胁RDT的疗效，世界卫生组织（WHO）已将这些缺失的监测作为优先事项。巢式PCR用于确认pfhrp 2缺失，但成本高且费力。由于副外显子pfhrp 3的假扩增，巢式外显子1 PCR产物的身份必须通过测序来确认。在这里，我们描述了一个新的一步PCR方法检测pfhrp 2。为了确定灵敏度和特异性，所有PCR均重复进行三次。以光诱导电子转移（PET）PCR检测18 srRNA为真阳性，一步法的灵敏度可达95.0%（88.7-98.4%）与巢式外显子1，99.0%（94.6-99.9%）和巢式外显子2，98.0%（93.0-99.8%），与巢式外显子1 100.0%（79.4-100.0%）和巢式外显子2 100.0%（74.4 - 100.0%）的特异性93.8%（69.8-99.8%）相当。测序结果显示，一步PCR不能扩增pfhrp 3。用于测量临床分离株中一步PCR的95%检测水平的逻辑回归模型提供了全血（WB）样本的133 p/mu L（95%置信区间（CI）：3- 793 p/mu L）估计值和干血斑（DBS）的385 p/mu L（95% CI：31-2133 p/mu L）估计值。当考虑协议属性时，一步PCR更便宜、更快并且更适合于高通量。总之，我们已经开发出一种更准确的PCR方法，可能是理想的应用世界卫生组织的协议调查pfhrp 2缺失症状的个人提出的卫生保健设施。
Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photoinduced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/mu L (95% confidence interval (CI): 3-793p/mu L) for whole blood (WB) samples and 385p/mu L (95% CI: 31-2133 p/mu L) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities.