Enhancement of loop mediated isothermal amplification's sensitivity and speed by multiple inner primers for more efficient identification of Vibrio parahaemolyticus

The modified loop-mediated isothermal amplification (LAMP), called multiple hybrid, inner primers (MHP)-LAMP, was developed to enhance the efficiency of the existing LAMP-based assay for Vibrio parahaemolyticus detection. The method was built on a conventional LAMP assay by employing 2 newly designed extra sets of primers to increase the initial binding sites of core primers on the V. parahaemolyticus’s rpoD gene from 8 to 12. With this strategy, the assay detection sensitivity was increased by 10 folds, with the detection limit (DL) approaching 100 copies of purified target genomic DNA (gDNA) as analyzed by real-time turbidity measurement and gel electrophoresis. The MHP also accelerated the rate of DNA amplification by 30%, rendering the assay faster. The MHP-LAMP assay did not cross- react with other pathogens, indicating that it was highly specific for V. parahaemolyticus detection. Whilst V. parahaemolyticus was used as a study model herein, our idea of using MHP to maximize assay sensitivity and speed is considered as a universal strategy that can be applied to enhance efficiency of LAMP-based assays for detecting any DNA and RNA of interest. • The strategy of using multiple hybrid, inner primers (MHP) to enhance LAMP assay's efficiency was demonstrated with success. • The MHP enhanced the sensitivity and speed of the existing LAMP assay, designed to detect V. parahaemolyticus, by 10 times and 30%, respectively. • The proposed strategy can be applied to boost up any other LAMP-based assay's diagnostic performance

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera

Potential utility of α,β-dehydrolovastatin (DHLV) as an anti-diarrheal agent for secretory diarrheas.

(A) Effect of DHLV on cholera toxin (CT)- and heat-stable toxin (STa)-stimulated Cl- secretion in T84 cell monolayers determined by ISC analysis. After apical addition of CT (1 μg/ml) or STa (100 μM) to stimulate the increasing of ISC, DHLV was added in both apical and basolateral solution. Representative ISC tracings are shown. (B) Effect of DHLV on CT-induced intestinal fluid secretion determined by ileal closed-loop weight/length ratios. Ileal closed-loops were injected with PBS (control) or PBS containing CT (μg/loop) with or without intraluminal (i.l.) and intraperitoneal (i.p.) administrations of DHLV (20 μM and 2 mg/kg, respectively). Representative photographs of ileal loops are shown. Summary of data are expressed as means of ileal closed-loop weight/length ratio ± S.E.M. (n = 5–6). *** p p n = 6–7). * p < 0.05 compared with control at 1 min; NS, non-significant compared with control at the same time point (one-way ANOVA).</p

No involvement of CFTR negative regulators in CFTR inhibition by DHLV.

(A) Schematic diagrams showing the regulatory mechanism of CFTR Cl- channel activity. (B) apical Cl- current (ICl-) tracing showing the effect of α,β-dehydrolovastatin (DHLV) on forskolin-induced CFTR Cl- secretion in T84 cells pre-treatment with compound C (50 μM) or and NaF plus Na3VO4 (1 mM) for 15 min. (C) Summary of concentration-inhibition studies. Data are fitted to Hill’s equation and expressed as means of % agonist-stimulated ICl- ± S.E.M. (n = 5–9).</p

Effect of statin derivatives isolated from soil fungus <i>Aspergillus sclerotiorum</i> and statin drugs on cAMP-dependent Cl^- secretion in T84 cell monolayers.

(A) Effect of α,β-dehydrolovastatin (DHLV), lovastatin, α,β-dehydrodihydromonacolin K, mevastatin, simvastatin, and pravastatin on cAMP-dependent Cl- secretion determined by ISC analysis. (B) Effect of CFTRinh-172 (CFTR inhibitor) on cAMP-dependent Cl- secretion determined by ISC analysis. All of compounds were added accumulatively in both apical and basolateral solutions at the indicated concentrations. Representative ISC tracings are shown. (C) Summary of concentration- inhibition studies. Data are fitted to Hill’s equation and expressed as means of % forskolin-stimulated ISC ± S.E.M. (n = 3–7).</p

End-point rapid detection of total and pathogenic Vibrio parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) in raw seafood using a colorimetric loop-mediated isothermal amplification-xylenol orange technique

Background Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis in humans worldwide. To ensure seafood safety and to minimize the occurrence of seafood-borne diseases, early detection of total V. parahaemolyticus (pathogenic and non-pathogenic strains) and pathogenic V. parahaemolyticus (tdh+ and/or trh1+ and/or trh2+) is required. This study further improved a loop-mediated isothermal amplification (LAMP) assay using xylenol orange (XO), a pH sensitive dye, to transform conventional LAMP into a one-step colorimetric assay giving visible results to the naked eye. LAMP-XO targeted rpoD for species specificity and tdh, trh1, and trh2 for pathogenic strains. Multiple hybrid inner primers (MHP) of LAMP primers for rpoD detection to complement the main primer set previously reported were designed by our group to maximize sensitivity and speed. Methods Following the standard LAMP protocol, LAMP reaction temperature for rpoD, tdh, trh1, and trh2 detection was first determined using a turbidimeter. The acquired optimal temperature was subjected to optimize six parameters including dNTP mix, betaine, MgSO4, Bst 2.0 WarmStart DNA polymerase, reaction time and XO dye. The last parameter was done using a heat block. The color change of the LAMP-XO result from purple (negative) to yellow (positive) was monitored visually. The detection limits (DLs) of LAMP-XO using a 10-fold serial dilution of gDNA and spiked seafood samples were determined and compared with standard LAMP, PCR, and quantitative PCR (qPCR) assays. Subsequently, the LAMP-XO assay was validated with 102 raw seafood samples and the results were compared with PCR and qPCR assays. Results Under optimal conditions (65 °C for 75 min), rpoD-LAMP-XO and tdh-LAMP-XO showed detection sensitivity at 102 copies of gDNA/reaction, or 10 folds greater than trh1-LAMP-XO and trh2-LAMP-XO. This level of sensitivity was similar to that of standard LAMP, comparable to that of the gold standard qPCR, and 10-100 times higher than that of PCR. In spiked samples, rpoD-LAMP-XO, tdh-LAMP-XO, and trh2-LAMP-XO could detect V. parahaemolyticus at 1 CFU/2.5 g spiked shrimp. Of 102 seafood samples, LAMP-XO was significantly more sensitive than PCR (P < 0.05) for tdh and trh2 detection and not significantly different from qPCR for all genes determined. The reliability of tdh-LAMP-XO and trh2-LAMP-XO to detect pathogenic V. parahaemolyticus was at 94.4% and 100%, respectively. Conclusions To detect total and pathogenic V. parahaemolyticus, at least rpoD-LAMP-XO and trh2-LAMP-XO should be used, as both showed 100% sensitivity, specificity, and accuracy. With short turnaround time, ease, and reliability, LAMP-XO serves as a better alternative to PCR and qPCR for routine detection of V. parahaemolyticus in seafood. The concept of using a one-step LAMP-XO and MHP-LAMP to enhance efficiency of diagnostic performance of LAMP-based assays can be generally applied for detecting any gene of interest

Effects of α,β-dehydrolovastatin (DHLV) on Ca²⁺-activated Cl^- channels (CaCC), the intracellular Ca²⁺ levels induced by ATP and Na⁺/K⁺ ATPases activity.

(A, left) Representative apical ICl- tracing showing the effect of ATP (100 μM) activation after 15 min pre-treatment with CFTRinh-172 (5 μM) with vehicle (control) or DHLV (20 μM). (A, right) Summary of the data of peak ICl- expressed as mean of peak ICl- ± S.E.M. (n = 5). * p 2+ levels were analyzed from the fluo-8-based fluorescence assays. Fluo-8 fluorescence intensity were measured following vehicle (control) or DHLV (20 μM) together with the addition of ATP (100 μM) and end up with EGTA (3 mM). (B) Representative values of the fractional change in fluorescence intensity relative to baseline (ΔF/F°) are shown. (C) Summary of total AUC values after baseline correction presented as the area under the curve (AUC) ± S.E.M (n = 4). NS, non-significant (Student’s t test). (D, left) Ouabain-sensitive ISC tracing showing the effect of DHLV on Na+-K+ ATPase activity. After T84 cells were permeabilized at apical membrane, ouabain (1 mM) was added. (D, right) Summary of the data are expressed as mean of ouabain-sensitive ISC ± S.E.M. (n = 4). NS, non-significant (Student’s t test).</p

Mechanism of α,β-dehydrolovastatin (DHLV) actions on cAMP-dependent CFTR activity in intestinal epithelial T84 cell monolayers.

(A) Schematic diagrams showing the regulatory mechanism of CFTR Cl- channel activity. (B) Effect of DHLV on cAMP-dependent Cl- secretion determined by apical ICl- analysis induced by forskolin (20 μM), genistein (20 μM), or CPT-cAMP (100 μM). The involvement of phosphodiesterase (PDE) and multidrug-resistance 4 (MRP4) was determined by 15 min pre-treatment with IBMX (1 mM) or MK571 (20 μM), respectively, prior to forskolin treatment. Representative ISC tracings are shown. (C) Summary of concentration-inhibition studies. Data are fitted to Hill’s equation and expressed as means of % agonist-stimulated ICl- ± S.E.M. (n = 3–7). (D) Effect of DHLV on intracellular cAMP levels. T84 cells were pre-treated with vehicle (control) or DHLV (20 μM) for an hour. Intracellular cAMP levels were measured by cAMP parameter assay kits. Data are expressed as concentrations of cAMP ± S.E.M. (n = 3). NS, non-significant compared with indicated group.</p

Effect of α,β-dehydrolovastatin (DHLV) on cAMP-dependent Cl^- secretion, cell viability and barrier function in T84 cell monolayers.

(A, left) Polarity of inhibition by α,β-dehydrolovastatin (DHLV) on cAMP-dependent Cl- secretion determined by ISC analysis. DHLV (20 μM) was added into apical and basolateral solutions in a separate experiment. Representative ISC tracings are shown. (A, right) Summary of the data expressed as mean of ISC ± S.E.M. (n = 5). (B) Reversibility of inhibition of CFTR-mediated Cl- secretion by DHLV (2 μM) in T84 cell monolayers. After the inhibition of CFTR-mediated Cl- secretion was stabilized, the bathing solution containing forskolin and DHLV was removed. Both chambers were gently washed 5 times and bathing solution containing forskolin were re-filled into the chamber. At the end of experiment, CFTRinh-172 (20 μM) was added into the apical chamber. A representative tracing of 3 independent experiments is shown (n = 3) (C) Effect of DHLV on cell viability evaluated by MTT assays. T84 cells were treated with DHLV at the indicated concentrations for 6 or 24 h (upper, lower). Data are expressed as % of cell viability compared to control ± S.E.M. (n = 4–6). (D) Effect of DHLV on intestinal barrier function. FITC-dextran flux assays were performed after 6 h of incubation with DHLV (20 μM). EGTA (3 mM) was used as a positive control. Data are expressed as concentration of FITC-dextran ± S.E.M. (n = 5–6). NS, non-significant; **** p < 0.0001 compared with control.</p