Development of a loop-mediated isothermal amplification assay targeting lmo0753 gene for detection of Listeria monocytogenes in wastewater.

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.  monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.  monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl-1 of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.  monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.  monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.  monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.  monocytogenes in wastewater from treatment plants. The LAMP assay detects L.  monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.  monocytogenes detection.

Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U µl-1 ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis.

A rapid and visual loop-mediated isothermal amplification assay to detect Leifsonia xyli subsp. xyli targeting a transposase gene.

UNLABELLED: Leifsonia xyli subsp. xyli (Lxx), causal organism of ratoon stunt (RSD), does not produce any reliable internal or external symptoms on sugarcane. Its detection on a large scale is solely based on microscopic and serological methods. These methods require well-equipped laboratories, are time consuming and are not feasible for near-field detection of Lxx. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Lxx without the use of sophisticated equipment. To the best of our knowledge, this is the first report on the detection of Lxx in 30 min via an isothermal amplification method at 65°C. A transposase gene, ISLxx5, was used to design a set of six primers specifically targeting eight genomic sequences. The xylem sap was used as template, thus circumventing the need to isolate pure genomic DNA. The positive reactions were visually detected through a colour change of hydroxynaphthol blue (HNB) from violet to light blue, thus, eliminating the need for gel electrophoresis. The LAMP method was 10 times more sensitive than serological detection and as sensitive as immunofluorescence microscopy (IFM). The simplicity and sensitivity of the ISLxx5 LAMP assay makes it suitable for near-field diagnosis of RSD. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of Leifsonia xyli subsp. xyli (Lxx) on a large scale is based on serological assays such as evaporative-binding enzyme-linked immunoassay (EB-EIA). These methods are time consuming and require well-equipped laboratories. This study presents the development of a loop-mediated isothermal amplification (LAMP) assay which allows detection of Lxx in 30 min at 65°C, using xylem sap as the template. The assay requires minimal laboratory equipment and could be used at near farm conditions, thus saving time and money required to transfer samples from remote areas to diagnostic laboratories. The LAMP method shows potential as an alternative detection method for RSD.

Snare protein expression and adenoviral transfection of amphicrine AR42J.

The amphicrine AR42J acinar cell line is an excellent model to study both exocrine and neuroendocrine exocytotic mechanisms. As a first step toward this goal, we determined the specific isoforms of the v- and t-SNARE and Munc18 families expressed in these cells. In addition, we show that dexamethasone-induced differentiation toward the exocrine phenotype causes an upregulation of several of these proteins. AR42J is notoriously difficult to transfect, limiting its usefulness as a model. However, we have now overcome this obstacle by acheiving high efficiency expression of a beta-galactosidase reporter gene and truncated SNAP-25 gene using adenoviral infection techniques. The AR42J cells can now be used to pursue and elucidate the distinct functions of individual SNARE isoforms used in endocrine and exocrine secretion within a single cell line.

The vesicle-associated membrane protein family of proteins in rat pancreatic and parotid acinar cells.

BACKGROUND & AIMS: The vesicle-associated membrane protein (VAMP) family of proteins may play an important role in regulating enzyme secretion from pancreatic and parotid acini. The purpose of this study was to characterize the isoforms produced in pancreatic and parotid acini and determine their subcellular locations. METHODS: Using a battery of specific antisera and recombinant tetanus toxin light chain (which cleaves VAMP-2 and cellubrevin), the presence of each VAMP molecule in the acini was determined by immunoblotting of subcellular membrane fractions; their localization was determined by confocal immunofluorescence microscopy and immunogold electron microscopy. RESULTS: Both VAMP-2 and cellubrevin were present on both the zymogen granule membrane and plasma membrane. VAMP-1 was not present in the acinar cell but was found in the nerve endings innervating the acini. As expected, pancreatic acinar VAMP-2 and cellubrevin were sensitive to cleavage by recombinant tetanus toxin. CONCLUSIONS: VAMP-2 and cellubrevin may play integral roles in exocytosis of the pancreatic and parotid acinar cells, whereas VAMP-1 is restricted to nerves that innervate the acini and may function to modulate exocrine activity.

Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells.

Syntaxins are cytoplasmically oriented integral membrane soluble NEM-sensitive factor receptors (SNAREs; soluble NEM-sensitive factor attachment protein receptors) thought to serve as targets for the assembly of protein complexes important in regulating membrane fusion. The SNARE hypothesis predicts that the fidelity of vesicle traffic is controlled in part by the correct recognition of vesicle SNAREs with their cognate target SNARE partner. Here, we show that in the exocrine acinar cell of the pancreas, multiple syntaxin isoforms are expressed and that they appear to reside in distinct membrane compartments. Syntaxin 2 is restricted to the apical plasma membrane whereas syntaxin 4 is found most abundantly on the basolateral membranes. Surprisingly, syntaxin 3 was found to be localized to a vesicular compartment, the zymogen granule membrane. In addition, we show that these proteins are capable of specific interaction with vesicle SNARE proteins. Their nonoverlapping locations support the general principle of the SNARE hypothesis and provide new insights into the mechanisms of polarized secretion in epithelial cells.

Characterization of SNARE protein expression in beta cell lines and pancreatic islets.

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.