A simple and rapid method for detection of Goose Parvovirus in the field by loop-mediated isothermal amplification

Virology Journal A simple and rapid method for detection of Goose Parvovirus in the field by loop-mediated isothermal amplification Yang JinLong 0 2 Yang Rui 2 Cheng AnChun 0 1 Wang MingShu 0 1 Fu LiZhi 2 Yang SongQuan 2 Zhang SuHui 2 Yang Liu 2 Xu ZhiYong 3 0 Avian Diseases Research Center, College of Veterinary Medicine of Sichuan Agricultural University , Yaan 625014, Sichuan Province , China 1 Key Laboratory of Animal Diseases and Human Health of Sichuan Province , Yaan 625014, Sichuan Province , China 2 Chongqing Academy of Animal Science , Chongqing 402460, Chongqing , China 3 College of Animal Sciences, Henan Institute of Science and Technology , Xinxiang 453003, Henan Province , China Background: Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, in a worldwide scale, GPV severely affects geese production. The objective of this study is to develop a loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of GPV in the field. Results: A set of six specific primers was designed by targeting the GPV VP3 DNA. With Bst DNA polymerase large fragment, the target DNA could be amplified at 65C as early as 20 min of incubation in a simple water bath. A positive reaction was identified through the detection of the LAMP product by color change visible to the naked eye. The detection limit of the assay was 28 copies/l of plasmid pVP3, and with equal sensitivity and specificity to fluorescent quantitative real-time PCR (FQ-PCR). Conclusions: The high sensitivity, specificity, and simplicity, as well as the high throughput, make this method suitable for specific detection of GPV infection in both field conditions and laboratory settings. The utilization of complicated equipment and conduct of technical training on the GPV LAMP were not necessary. - Background Goose parvovirus (GPV) is a well known causative agent of Gosling plague (GP), an acute, contagious, and fatal disease referred to as Derzsys disease [1]. GPV has been formally classified as a member of the genus Dependovirus under the family, Parvoviridae [2]. It was first described as a clinical entity by Fang [3]. In the realm of research, GPV has attracted much attention owing to tremendous economic loss for countries engaged in industrialized goose production; the virus infection has spread rapidly worldwide, resulting in high rates of morbidity and mortality [1,4-6]. Several detection methods have been developed for identifying GPV, such as agar-gel diffusion precipitin test, virus neutralization (VN) assay, enzyme-linked immunosorbent assay (ELISA) [5], qualitative PCR [7,8], and fluorescent quantitative real-time PCR (FQ-PCR) [9]. All are effective and accurate in detecting the virus infection in laboratory settings, but they require the use of expensive equipment and are laborious and time-consuming. Thus, these methods are considered unfavorable for use on a large-scale basis. In contrast, a more preferred detection method would be one that is not only speedy and sensitive, but also simple and economical during practical applications [10]. Recently, a loop-mediated isothermal amplification (LAMP) reaction was developed as an alternative method to meet the abovementioned requirements. The LAMP method allows the whole reaction process, including denaturing, to proceed at a constant temperature by incubating the reagents in a simple incubator. As a specific nucleic acid amplification method, it can easily perform and amplify nucleic acid at isothermal conditions (i.e., 60-65 C) within 1 h of incubation [11-13]. LAMP reaction requires four or six primers based on six or eight distinct regions of the target DNA, hence allowing high degree of specificity during viral detection. The presence of amplified products can be detected at a short time. By the end of the reaction, the presence or absence of the target DNA can be judged visually by the appearance of a white precipitate of magnesium pyrophosphate, or a green color of the solution stained by SYBR green I. The presence of multiple bands of LAMP reaction products in agarose gel electrophoresis indicates a mixture composed of stemloop DNAs with various sizes of stem and cauliflower-like structures having multiple loops, which is induced by alternately annealing inverted repeats of the target sequence in the same strand [11,14]. In addition, the LAMP method does not require any special reagent or sophisticated temperature control device. Since it only needs simple equipment, cost-effective genetic tests can be easily achieved. Both simple detection and real-time detection of the reaction are deemed possible http://loopamp. eiken.co.jp/e/index.html. Specifically, the LAMP method has already been applied in the specific detection of animal viruses, such as hepatitis B virus [15], Japanese encephalitis viral [16], and H9 avian influenza virus [17]. However, to the best of our knowledge, no study has yet used the technique to detect GPV. In this study, we report the development of LAMP assay for the specific, rapid, and sensitive detection of GPV in infected goslings. Results Optimized LAMP reaction LAMP reaction was performed using plasmid (pVP3) DNA as template in order to determine optimal temperature and time of reaction. The amplicons were formed at 61, 62, 63, 64, and 65C and the clearest product was detected at 65C (Fig. 1A). Thus, 65C was used as the optimal temperature for the succeeding assays. Meanwhile, LAMP products were also detected as early as 20 min at 65C (Fig. 1B). Although wellformed bands in the system could be detected as early as 20 min, reaction time was optimized and set at 40 min to ensure positive detection of templates with lower concentration. Specificity of the LAMP assay The specificity of LAMP and FQ-PCR was tested using templates extracted from GPV and other viruses. Only GPV showed a positive reaction; no DNA band was observed from the other seven animal pathogens (Fig. 2). Results of FQ-PCR (data not shown) correlated well with the LAMP method [9], indicating that LAMP is as specific as FQ-PCR for GPV detection. Sensitivity of LAMP assay The detection limit of LAMP using plasmid DNA was set at 28 copies/l (Fig. 3). In comparison with the detection limit of FQ-PCR (date not shown) [9], LAMP was observed to similarly sensitive to the FQ-PCR system. Detection of LAMP products by naked eye observation LAMP products could also be detected with the naked eye by observing white turbidity in the reaction mixture (Fig. 4A) or by color change of the solution stained by SYBR Green I (Fig. 4B). As shown by Fig. 4A, white turbidity could be observed from products of the reaction with 2.8 102 to 2.8 1011 copies/l of plasmid, but not from the negative control and the 2.8 100 to 2.8 101 copies/l. In Fig. 4B, after the addition of 1 l of diluted SYBR Green I to the reaction tube, the color of the LAMP reaction solution changed from (...truncated)