Gigante Crystal M, Wicker Vaughn, Wilkins Kimberly, Seiders Melanie, Zhao Hui, Patel Puja, Orciari Lillian, Condori Rene Edgar, Dettinger Lisa, Yager Pamela, Xia Dongxiang, Li Yu
Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA.
Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA.
J Virol Methods. 2025 Apr;333:115070. doi: 10.1016/j.jviromet.2024.115070. Epub 2024 Nov 21.
Reliable, validated diagnostic tests are critical for rabies control in animals and prevention in people. We present a performance assessment and updates to the LN34 real-time RT-PCR assay for rabies diagnosis in postmortem animal brain samples. In two U.S. laboratories during 2017-2022, routine used of the LN34 assay produced excellent diagnostic sensitivity (99.72-100 %) and specificity (99.99-100 %) compared to the direct fluorescence antibody test (DFA). Almost all (>90 %) DFA indeterminate results caused by non-specific or atypical fluorescence were negative by LN34 testing, representing up to 111 cases where unnecessary post-exposure prophylaxis could be avoided. LN34 assay original primer sequences showed low sensitivity for some rare lyssaviruses. Increased primer concentration combined with new primer formulation showed improved performance for impacted lyssaviruses with no loss in performance across diverse rabies virus variants from clinical samples. The updated LN34 and internal control assays were combined into a single-well LN34 multiplexed (LN34M) format, run at half reagent volumes. The LN34M assay showed similar detection of rabies virus to the singleplexed assay with simplified assay set-up, lower cost, and improved quality controls.
可靠、经过验证的诊断测试对于动物狂犬病控制和人类预防至关重要。我们展示了用于死后动物脑样本狂犬病诊断的LN34实时逆转录聚合酶链反应（RT-PCR）检测方法的性能评估及更新。在2017年至2022年期间，美国的两个实验室中，与直接荧光抗体检测（DFA）相比，常规使用的LN34检测方法具有出色的诊断敏感性（99.72 - 100%）和特异性（99.99 - 100%）。几乎所有（>90%）由非特异性或非典型荧光导致的DFA不确定结果经LN34检测均为阴性，这意味着多达111例可避免不必要的暴露后预防情况。LN34检测方法的原始引物序列对一些罕见的狂犬病病毒敏感性较低。提高引物浓度并结合新的引物配方，对受影响的狂犬病病毒表现出更好的性能，且对临床样本中各种狂犬病病毒变体的检测性能没有损失。更新后的LN34和内部对照检测方法被整合到一个单孔LN34多重检测（LN34M）形式中，以一半的试剂体积运行。LN34M检测方法对狂犬病病毒的检测效果与单重检测相似，同时简化了检测设置，降低了成本，并改进了质量控制。
