Krevolin Mark D, Hardy David, Pane Jim, Aslam Shagufta, Behrens Catherine M
Roche Molecular Systems, Pleasanton, California.
Roche Molecular Systems, Pleasanton, California.
J Mol Diagn. 2017 Mar;19(2):288-294. doi: 10.1016/j.jmoldx.2016.10.003. Epub 2016 Dec 30.
The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (C) values for cobas HPV detection were evaluated over time and various temperatures, and mean C differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) C differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: -2.58 (-3.16 to -2.01), -2.63 (-3.62 to -1.64), and -3.39 (-4.95 to -1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) C differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: -0.48 (-0.98 to 0.02) and -0.23 (-0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [-0.35 (-0.57 to -0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath.
在含有福尔马林的介质中，核酸与蛋白质之间形成化学交联，这给在SurePath保存液中收集的宫颈样本进行人乳头瘤病毒（HPV）检测带来了挑战。开发了一种分析前处理方法，即加入亲核缓冲液并将样本加热至120°C，以逆转交联效应，并提高SurePath中cobas HPV检测的核酸可及性。随时间和不同温度评估了cobas HPV检测的循环阈值（C）值，并评估了预处理的SurePath样本与未处理的SurePath样本以及在PreservCyt中收集的样本之间的平均C差异。未经预处理时，7天后HPV的低病毒水平（1×检测限）就不再能检测到。对于前瞻性收集的标本，预处理样本与未处理样本之间的平均（95%置信区间）C差异表明，在所有处理样本类别中HPV DNA回收率均有所提高：对于其他12种高危HPV类型、HPV16和HPV18，分别为-2.58（-3.16至-2.01）、-2.63（-3.62至-1.64）和-3.39（-4.95至-1.82）。此外，预处理的SurePath样本的平均（95%置信区间）C差异与同时收集的PreservCyt样本相当：对于HPV16和HPV18，分别为-0.48（-0.98至0.02）和-0.23（-0.93至0.46）；对于其他12种高危HPV类型，观察到边缘显著差异[-0.35（-0.57至-0.13）]。因此，这种分析前处理程序确保了在SurePath中进行cobas HPV检测的一种经过验证、安全且准确的方法。
