Martínez Miguel A, Soto-Del Río María de Los Dolores, Gutiérrez Rosa María, Chiu Charles Y, Greninger Alexander L, Contreras Juan Francisco, López Susana, Arias Carlos F, Isa Pavel
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico.
Departamento de Microbiología Molecular, Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico.
J Clin Microbiol. 2015 Jan;53(1):136-45. doi: 10.1128/JCM.01317-14. Epub 2014 Oct 29.
Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals.
肠胃炎是人类和其他动物的一种临床疾病，其特征为呕吐和腹泻，由多种病原体引起，包括病毒。随着新分子工具的开发，越来越多的病毒种类与肠胃炎相关或在粪便样本中被发现。在这项研究中，开发并测试了一种理论上能够并行检测100多种病毒种类的DNA微阵列。最初用10种不同病毒种类进行了验证，另外5种病毒种类使用临床样本进行了验证。确定了人腺病毒C（HAdV）、人星状病毒（HAstV）和A组轮状病毒（RV-A）的检测限为1×10³个病毒颗粒。此外，当添加外源RNA时，RV-A的检测限降低了一个对数级。在一小群肠胃炎患儿的临床样本（n = 76）中，微阵列在92%的样本中检测到至少一种病毒种类。63个样本（83%）被鉴定为单一感染，7个样本（9%）被鉴定为多种病毒的混合感染。最常见的病毒种类是RV-A（58%），其次是环病毒（15.8%）、HAstV（6.6%）、HAdV（5.3%）、诺沃克病毒（6.6%）、人肠道病毒（HEV）（9.2%）、人细小病毒（1.3%）、札幌病毒（1.3%）和人博卡病毒（1.3%）。为了进一步测试微阵列的特异性和敏感性，通过逆转录PCR（RT-PCR）检测5种胃肠道病毒对结果进行了验证。RT-PCR检测在59个样本（78%）中检测到病毒。微阵列在检测RV-A、HAstV和杯状病毒方面表现良好，而对HAdV和HEV的敏感性较低。此外，在混合感染的检测中观察到一些差异，并通过对相关病毒的逆转录定量PCR（RT-qPCR）解决。观察到遗传物质数量的差异有利于检测最丰富的病毒。这项研究中描述的微阵列应有助于了解人类和动物肠胃炎的病因。
