ABSTRACT
Maize chlorotic mottle virus (MCMV), an important quarantine virus, causes lethal necrosis in maize when coinfected with a potyvirid, which is seriously threatening the production of maize worldwide. In this study, recombinase polymerase amplification (RPA), a novel isothermal DNA amplification and detection technique, was developed to detect MCMV in maize crops. A pair of specific primers was designed based on the conserved sequences of the MCMV coat protein region. The RT-RPA assay was carried out as an isothermal reaction at 38 °C that was complete within 30 min, and no cross-reactivity was detected with other viruses infecting maize in China. The limit of detection of the RT-RPA assay was tenfold lower than that of ordinary RT-PCR. Moreover, this method was successfully applied to test field-collected samples. The newly developed RT-RPA assay offers a reliable, sensitive and efficient method for rapid detection of MCMV in maize in equipment-limited diagnostic laboratories and on-site facilities.
Subject(s)
Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Plant Diseases/virology , Tombusviridae/isolation & purification , Capsid Proteins/genetics , China , DNA Primers/genetics , Sensitivity and Specificity , Temperature , Time Factors , Tombusviridae/classification , Tombusviridae/geneticsABSTRACT
Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is an important quarantine plant virus worldwide, and often causes seriously damages to productions of watermelon, melon, cucumber and other cucurbit crops. In this study, we developed a novel isothermal recombinase polymerase amplification (RPA) technique for detection of CGMMV in watermelon samples. A pair of CGMMV specific RPA primers was prepared based on the conserved CGMMV coat protein gene sequences. The result showed that this RPA detection method can be performed at 38⯰C and completed in about 30â¯min, and there was no cross-reactivity with other common cucurbit viruses. Sensitivity assay showed that this RPA method was more sensitive compared with the regular RT-PCR. Using field-collected watermelon tissue samples, we have demonstrated that this newly developed method is rapid, easy to use and reliable for CGMMV detection, especially in resource-limited laboratories or on-site facilities.
Subject(s)
Citrullus/virology , Nucleic Acid Amplification Techniques/methods , Plant Diseases/virology , Tobamovirus/isolation & purification , DNA Primers , Recombinases/genetics , Sensitivity and Specificity , Tobamovirus/geneticsABSTRACT
Laser scanning confocal microscope (LSCM) is currently the only equipment to observe fluorescence. However, this technique has disadvantages such as high cost and long test process. In this study, we developed a new system of laser-induced fluorescence (LIF) for microfluidic chip applied to detecting the expression of green fluorescent protein (GFP) in Bacillus subtilis. This novel system was comprised of laser device, optics unit, microfluidic chip, photomultiplier and computer treatment unit. The tests indicated that microfluidic chip could detect the expression of GFP as sensitively as LSCM in Bacillus subtilis. Moreover, this LIF detection system could instead of PCR to identify the positive clone in this special case. Nevertheless, the LIF system only was suitable to detect the fluorescent strength of GFP, and could not meet the request of some cases for example protein location. Therefore, this system will be applied in environmental detection with microbe, drug discovery and other cases.
Subject(s)
Bacillus subtilis/isolation & purification , Bacillus subtilis/metabolism , Green Fluorescent Proteins/biosynthesis , Microfluidic Analytical Techniques/methods , Green Fluorescent Proteins/geneticsABSTRACT
The SRP receptor FtsY, which is involved in targeting and translocating membrane protein, is generally composed of the N-terminal domain and the NG domain. Although FtsY was highly homologous in the composition of amino acids and functions among microbes, the different mechanism in the location of FtsYs from different bacteria such as S. coelicolor and E. coli were discovered in this study by laser scanning confocal microscope (LSCM) in vivo and molecular techniques in vitro. The results revealed that the N-terminal domain of S. coelicolor FtsY was indispensable for FtsY's anchoring membrane, and while the A domain of E. coli FtsY was dispensable. Moreover, the A domain of E. coli FtsY might promote itself to bind the membrane depending on the location images and Western blotting.
