Vernick K D, Keister D B, Toure A, Toure Y T
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Am J Trop Med Hyg. 1996 Apr;54(4):430-8. doi: 10.4269/ajtmh.1996.54.430.
We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed.
我们使用恶性疟原虫子孢子生殖阶段的小亚基核糖体RNA（SSU rRNA）特异性序列，设计了一种逆转录聚合酶链反应（RT-PCR）检测方法，该方法能够在从潜在感染蚊子中纯化的总RNA中检测到0.1个子孢子。我们制备了一种合成RNA，它在RT-PCR中可被与寄生虫SSU rRNA相同的引物扩增，可作为内部对照和竞争定量标准。我们通过用来自纯化和计数的子孢子的RNA制作标准曲线，对该子孢子定量检测方法进行了校准。该检测方法在单次反应中能准确测量子孢子数量，线性范围至少为三个数量级。文中还讨论了该检测方法的一些应用及局限性。
