Development and evaluation of a loop-mediated isothermal amplification assay for the rapid detection of porcine cytomegalovirus under field conditions

Jin-Long Yang,Su-Hui Zhang,Zuo-Hua Liu,Ming Wen,Rui Yang,Yong Huang

doi:10.1186/1743-422x-9-321

Abstract

BackgroundPorcine cytomegalovirus (PCMV) induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets. This study aimed to develop a new loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of PCMV under field conditions.MethodsTissue obtained from nine-week-old PCMV-free Landrace pigs or pig samples from postmortem examinations were analyzed. The samples were found to have clinical signs and lesions consistent with inclusion body rhinitis. Six specific primers were designed by targeting the PCMV DNA polymerase (DPOL) DNA. The LAMP reaction was optimized in a water bath. The sensitivity and specificity of LAMP and polymerase chain reaction (PCR) were compared.ResultsPCMV DNA was amplified at 65°C, and the result could be detected as early as 30 min into the reaction. Positive reactions could be visualized by the naked eye as a color change brought on by the addition of SYBR Green. The sensitivity and specificity of LAMP were found to be similar to those of the PCR.ConclusionsLAMP is a high-throughput technique for the detection of PCMV and has a high specificity, sensitivity and simplicity; these factors make it suitable for detection of PCMV under field conditions.

Highlights

Porcine cytomegalovirus (PCMV) induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets
polymerase chain reaction (PCR) can be applied for detecting PCMV infection, and it is considered more useful than other techniques for the prediction of PCMV [18]
loop-mediated isothermal amplification (LAMP) products were detected in the reaction mixture at 65°C within as little as 30 min (Figure 1B)

Summary

Introduction

Porcine cytomegalovirus (PCMV) induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets. Porcine cytomegalovirus (PCMV) is a β-herpesvirus that was first described in 1955 [1] It shares sequence homology with human herpesviruses 6 and 7 and human cytomegalovirus (HCMV) [2,3,4,5,6]. It usually induces silent infection in adult pigs but often a fatal, generalized infection in newborn piglets, with 90% of pigs in the UK being seropositive [3,7]. PCR can be applied for detecting PCMV infection, and it is considered more useful than other techniques for the prediction of PCMV [18]

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