Abstract
Large-scale Hand, Foot, and Mouth Disease (HFMD) outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs). Among them, human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1–2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells). The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11) by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.
Highlights
Hand, Foot, and Mouth Disease (HFMD) is a common illness of infants and children under 10 years of age, and is characterized by fever, sores in mouth, and rash with blisters
The viral RNA of Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) derived from virus stocks of known titer was 10-fold serially diluted and used to determine the detection limits of the two real-time RT-PCR assays
The results demonstrated that the sensitivity was 0.1 TCID50/ml for HEV71 and 0.1 TCID50/ml for CVA16 by multiplex real-time RT-PCR assay, and 0.1 TCID50/ml for human enteroviruses (HEVs) in a broad-spectrum real-time RT-PCR assay
Summary
Foot, and Mouth Disease (HFMD) is a common illness of infants and children under 10 years of age, and is characterized by fever, sores in mouth, and rash with blisters. In China, the number of HFMD cases and fatal HFMD cases has been increasing every year, and multiple major HFMD outbreaks caused by Human enterovirus 71 (HEV71) had occurred in several provinces in China. Timely detection and differentiation of HEV71 from CVA16 in the early phase of HFMD infection are important, which is helpful to reduce the deaths caused by HEV71. Real-time RT-PCR assay has gained wider acceptance for viral diagnosis in laboratories because of high sensitivity and specificity as well as rapid detection. It provides real-time monitoring of the amplification process through fluorescence emission [10]
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