Rapid and Sensitive Detection of Singapore grouper iridovirus by Loop-Mediated Isothermal Amplification【精品论文大全】 .doc
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1、精品论文推荐Rapid and Sensitive Detection of Singapore grouper iridovirus by Loop-Mediated Isothermal AmplificationMao Xinliang 1,Zhou Sheng1,Xu Dan,Gong Jie,Cui Huachun,Qin Qiwei *State Key Laboratory of Biocontrol,College of Life Science,Sun Yat-sen University,Guangzhou(510275)E-mail:AbstractAims: The a
2、im of this paper is to develop a loop-mediated isothermal amplification (LAMP) methodfor rapid, sensitive and inexpensive detection of SGIV in grouper, Epinephelus sp.Methods and Results: A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragmen
3、t, the target DNA can be amplified as early as 20 min at 65C in a simple water bath. The detection limit is about 0.02 fg (equivalent to 6.3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross reactions with 7 other aquatic animal viruses indi
4、cating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus infected GP cells and grouper tissues effectively. Conclusions: The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for detection of SGIV in cells and in grouper tissues.Significa
5、nce and Impact of the Study: The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.Keywords:SGIV;LAMP;Grouper;Nested PCR.1. IntroductionIridoviruses are icosahedral deoxyriboviruses with a large double-stranded DN
6、A genome that can be categorized into five genera (Williams et al., 2005). In recent years, iridoviruses are well known as causative agents of serious systemic diseases among feral, cultured food and ornamental fish, and have been identified from more than 100 fish species in the last decade worldwi
7、de (Piaskoski and Plumb,1999; Hyatt et al., 2000; Wang et al., 2007). These viruses have attracted much attention because of their ecological impacts and caused tremendous economic losses in the aquaculture industry in various parts of the world (Langdon et al., 1986; Ahne et al., 1989; Inouye et al
8、., 1992; Pozet et al., 1992; Bloch and Larsen, 1993; Kasornchandra and Khongpradit, 1995; Plumb et al., 1996; Tapiovaara et al.,1998; Qin et al., 2003). Grouper, Epinephelus spp., the major species being maricultured in China and Southeast Asian countries, are high priced and popular seafood fish. I
9、n recent years, with the rapidly developing marine farming activities, outbreaks of iridoviral diseases have affected severely many highly valued species such as grouper causing heavy economic losses (Chua et al., 1994; Chou et al.,1998). Recently, a pathogenic iridovirus was isolated from diseased
10、brown-spotted grouper, Epinephelus tauvina and Malabar grouper, Epinephelus malabaricus in our laboratory using the method of cell culture isolation. The virus caused more than 90% mortality in cultured grouper, and could induce an advanced cytopathic effect (CPE) in a grouper cell line (GP). The di
11、seased fish spleens enlarged with hemorrhage, and multifocal areas of splenic degeneration were observed. Larege round basophilic cells with displaced nuclei were detected predominantly in spleen and kidney (Qin et al.,2002). The virus has been characterised as a new species of the genus Ranairus, f
12、amily Iridoiridae based on morphological, biochemical and genetic properties and named as Singapore grouper iridovirus (SGIV) (Qin et al., 2001, 2003).Several detection methods have been developed to detect the presence of SGIV in cell culture and in the cultured grouper ranging from electron micros
13、copic observations (Qin et al., 2001),1These authors contribute equally to this work.- 13 -immunofluorescence antibody test (IFAT) and antigen-capture enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies (Qin et al., 2002; Shi et al., 2003), in situ hybridization (Hua
14、ng et al., 2004), flow cytometry (FCM) (Qin et al., 2005) and a bead-based microfluidic device (Liu et al., 2005). All these methods are effective and accurate in detecting the virus infection in laboratory. However, these methods have some intrinsic disadvantages such as the requirement for expensi
15、ve equipments and reagents, or laborious and time consuming, rendering their unfavorable for use on a wide-scale basis. Detection method not only rapid and sensitive but also simple and economical to handle is needed for practical application.To meet these requirements, a loop-mediated isothermal am
16、plification (LAMP) reaction was developed as an alternative method. LAMP is a specific nucleic acid amplification method, which is easy to perform and can amplify nucleic acid at isothermal conditions at 6065 C within 1 h incubation period (Notomi et al., 2000; Mori et al., 2001; Nagamine et al., 20
17、02). The LAMP reaction requires four or six primers, which are based on six or eight distinct regions of the target DNA, hence it allows for a high degree of specificity for viral detection. At the end of the reaction, the presence or absence of the target DNA is judged visually by the appearance of
18、 a white precipitate of magnesium pyrophosphate or green color of the solution stained by SYBR green I. The presence of multiple bands of the LAMP reaction products in agarose gel electrophoresis indicative of a mixture composed of stem-loop DNAs with various sizes of stem and cauliflower-like struc
19、tures with multiple loops induced by annealing between alternatively inverted repeats of the target sequence in the same strand (Notomi et al., 2000; Nagamine, et al., 2002). LAMP has been applied for specific detection of aquatic animal viruses such as white spot syndrome virus (WSSV) (Tomoya et al
20、., 2004), red sea bream iridovirus (RSIV) (Christopher et al.,2004), koi herpes virus (KHV) (Hatem and Mansour, 2005) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Sun et al., 2006). In this study, the LAMP for specific, rapid and sensitive detection of SGIV in infected cell cu
21、ltures and fish was developed.2. Materials and methods2.1 Cell cultures and virus infectionThe GP cells, a permanent marine fish cell line, derived from embryos of grouper, E. tauvina (Chew-Lim et al., 1994), were cultured in 75 cm2 flasks at 25 C in Eagles minimum essential medium (EMEM)(Sigma,St.L
22、ouis,MO)supplementedwith5mMN-2-hydroxyethyl piperazine-N-2-ethanesulfornc acid (HEPES) buffer (Sigma), 100 IU ml1 penicillin, 100 g ml1 streptomycin, 0.116 M sodium chloride, 7% sodium bicarbonate, and 10% fetal calf serum (Sigma). The iridovirus used in this study is SGIV, strain of A3/12/98 PPD is
23、olated from diseased groupers, Epinephelus tauvina and E. malabaricus in Singapore (Qin et al., 2003). The GP cell culture monolayers were infected with virus at a multiplicity of infection (MOI) of approximately 0.1. When advanced cytopathic effect (CPE) was appeared, the virus-infected cells were
24、harvested and stored at-20 C for DNA extraction.For artificially infection, grouper in average weight of 60g were maintained in 500 L tanks (at 25 C)filled with air-pumped circulating sea water. Each fish was injected intraperitoneally with 100l 105TCID50 ml1 of SGIV. The organs (spleen, kidney, liv
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