Honma Shinjiro, Chizhikov Vladimir, Santos Norma, Tatsumi Masatoshi, Timenetsky Maria do Carmo S T, Linhares Alexandre C, Mascarenhas Joana D'Arc P, Ushijima Hiroshi, Armah George E, Gentsch Jon R, Hoshino Yasutaka
Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-8026, USA.
J Clin Microbiol. 2007 Aug;45(8):2641-8. doi: 10.1128/JCM.00736-07. Epub 2007 Jun 13.
Previously, we reported the development of a microarray-based method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) (V. Chizhikov et al., J. Clin. Microbiol. 40:2398-2407, 2002). The expanded version of the rotavirus microarray assay presented herein is capable of identifying (i) five clinically relevant human rotavirus VP4 genotypes (P[4], P[6], P[8], P[9], and P[14]) and (ii) five additional human rotavirus VP7 genotypes (G5, G6, G8, G10, and G12) on one chip. Initially, a total of 80 cell culture-adapted human and animal reference rotavirus strains of known P (P[1] to P[12], P[14], P[16], and P[20]) and G (G1-6, G8 to G12, and G14) genotypes isolated in various parts of the world were employed to evaluate the new microarray assay. All rotavirus strains bearing P[4], P[6], P[8], P[9], or P[14] and/or G1 to G6, G8 to G10, or G12 specificity were identified correctly. In addition, cross-reactivity to viruses of genotype G11, G13, or G14 or P[1] to P[3], P[5], P[7], P[10] to P[12], P[16], or P[20] was not observed. Next, we analyzed a total of 128 rotavirus-positive human stool samples collected in three countries (Brazil, Ghana, and the United States) by this assay and validated its usefulness. The results of this study showed that the assay was sensitive and specific and capable of unambiguously discriminating mixed rotavirus infections from nonspecific cross-reactivity; the inability to discriminate mixed infections from nonspecific cross-reactivity is one of the inherent shortcomings of traditional multiplex reverse transcription-PCR genotyping. Moreover, because the hybridization patterns exhibited by rotavirus strains of different genotypes can vary, this method may be ideal for analyzing the genetic polymorphisms of the VP7 or VP4 genes of rotaviruses.
此前，我们报道了一种基于微阵列的方法，用于鉴定五种临床相关的G基因型（G1至G4和G9）（V. Chizhikov等人，《临床微生物学杂志》40:2398 - 2407，2002年）。本文介绍的轮状病毒微阵列检测的扩展版本能够在一张芯片上鉴定：（i）五种临床相关的人类轮状病毒VP4基因型（P[4]、P[6]、P[8]、P[9]和P[14]），以及（ii）另外五种人类轮状病毒VP7基因型（G5、G6、G8、G10和G12）。最初，总共使用了80株已知P（P[1]至P[12]、P[14]、P[16]和P[20]）和G（G1 - 6、G8至G12以及G14）基因型的细胞培养适应型人类和动物参考轮状病毒株来评估这种新的微阵列检测方法。所有携带P[4]、P[6]、P[8]、P[9]或P[14]以及/或者G1至G6、G8至G10或G12特异性的轮状病毒株均被正确鉴定。此外，未观察到与G11、G13或G14基因型病毒或P[1]至P[3]、P[5]、P[7]、P[10]至P[12]、P[16]或P[20]的交叉反应。接下来，我们通过该检测方法分析了在三个国家（巴西、加纳和美国）收集的总共128份轮状病毒阳性人类粪便样本，并验证了其有效性。这项研究的结果表明，该检测方法灵敏且特异，能够明确区分混合轮状病毒感染与非特异性交叉反应；无法区分混合感染与非特异性交叉反应是传统多重逆转录 - PCR基因分型的固有缺点之一。此外，由于不同基因型的轮状病毒株表现出的杂交模式可能不同，这种方法可能是分析轮状病毒VP7或VP4基因遗传多态性的理想方法。
