TaqMan MGB Probe Fluorescence Real-Time Quantitative PCR for Rapid Detection of Chinese Sacbrood Virus

Li Yongfei,Liu Jinhua,Song Yingjin,Wenzhe Ho,Ma Mingxiao,Li Li

doi:10.1371/journal.pone.0052670

Abstract

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

Highlights

Sacbrood virus (SBV) is a picorna-like virus that affects the honey bee (Apis mellifera) and results in the death of the larvae [1]
This paper describes a TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR assay to quantify Chinese SBV (CSBV)
No amplification was detected when these TaqMan PCR conditions were performed on Complementary DNA (cDNA) obtained from chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV), black queen cell virus (BQCV) or deformed wing virus (DWV) samples (Fig. 3)

Summary

Introduction

Sacbrood virus (SBV) is a picorna-like virus that affects the honey bee (Apis mellifera) and results in the death of the larvae [1]. Infected larvae change in color from white to pale yellow and die. After death they dry out and form a distinctive dark brown gondola-shaped scale [2]. SBV causes a fatal infection in bee larvae, but may infect the adult bee and infected workers may have decreased life spans [3,4]. When this infection occurs in adults, obvious physical signs of disease are lacking [3,5]SBV infection occurs most frequently in the spring and this timing is believed to reflect the availability of susceptible larvae and young adults that are at their height in this season when the colony is growing most rapidly [3]

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Conclusion