Evaluation of loop-mediated isothermal amplification assay for visual detection of Acinetobacter baumannii directly from soil and water sample from Mangalore.

Acinetobacter baumannii is a well-known nosocomial pathogen that commonly inhabits soil and water and has been implicated in numerous hospital-acquired infections. The existing methods for detecting A. baumannii have several drawbacks, such as being time-consuming, expensive, labor-intensive, and unable to distinguish between closely related Acinetobacter species. Thus, it is important to have a simple, rapid, sensitive, and specific method for its detection. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue dye to visualize A. baumannii by targeting its pgaD gene. The LAMP assay was performed using a simple dry bath and was shown to be specific and highly sensitive, as it could detect up to 10 pg/µl of A. baumannii DNA. Further, the optimized assay was used to detect A. baumannii in soil and water samples by culture-medium enrichment. Out of 27 samples tested, 14 (51.85%) samples were positive for A. baumannii through LAMP assay, while only 5 (18.51%) samples were found to be positive through conventional methods. Thus, the LAMP assay has been found to be a simple, rapid, sensitive, and specific method that can be used as a point-of-care diagnostic tool for detecting A. baumannii.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Effect of sub-minimum inhibitory concentration of ceftriaxone on the expression of outer membrane proteins in Salmonella enterica serovar Typhi.

Multi-drug resistance (MDR) in Salmonella is one of the major reasons for foodborne outbreaks worldwide. Decreased susceptibility of Salmonella Typhi to first-line drugs such as ceftriaxone, ciprofloxacin, and azithromycin has raised concern. Reduced outer membrane proteins (OMPs) permeability and increased efflux pump transportation are considered to be the main reasons for the emergence of antibiotic resistance in Salmonella. The present study aimed to assess the expression of OMPs at sub-lethal concentrations of ceftriaxone in S. Typhi (Sl5037/BC, and Sl05). The S. Typhi strains were exposed to sub-MIC and half of the sub-MIC concentrations of ceftriaxone at three different time intervals (0 min, 40 min, and 180 min) and analyzed for differential expression of OMPs. Further, the expression variation of OMP encoding genes (yaeT, ompX, lamb, ompA, and ybfM) in response to ceftriaxone was evaluated using real-time PCR. The genes like lamB, ompX, and yaeT showed significant downregulation (p < 0.05) compared to the control without antibiotic exposure, whereas ybfM and ompA showed a moderate downregulation. The expression of omp genes such as lamB, ompA, ompX, ybfM, and yaeT were found to be low in the presence of ceftriaxone, followed by time and dose-dependent. The study provides insights into the possible involvement of OMPs in drug resistance of S. Typhi, which could help develop a therapeutic strategy to combat MDR isolates of S. Typhi.

Isothermal amplification-based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments.

The coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, the virus has been detected in 219 countries of the world. Therefore, managing the disease becomes the priority, in which detecting the presence of the virus is a crucial step. Presently, real-time RT polymerase chain reaction (RT-qPCR) is considered a gold standard nucleic acid amplification test (NAAT). The test protocol of RT-qPCR is complicated, places high demands on equipment, testing reagents, research personnel skills and is expensive. Therefore, simpler point-of-care (POC) tests are needed to accelerate clinical decision-making and take some of the workload from centralized test laboratories. Various isothermal amplification-based assays have been developed for the sensitive detection of different microorganisms, and recently some of them have been applied for detection of SARS-CoV-2. These do not require any programable thermocycler, can produce the results in a single temperature, and therefore, are considered simple. Unlike RT-qPCR, these methods are highly sensitive, specific, less time-consuming, simple and affordable, and can be used as POC diagnostic kit for COVID-19. In this review, we have discussed the potential of isothermal amplification-based assays as an alternative to RT-qPCR for the detection of SARS-CoV-2.

Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Rapid visual detection of Vibrio parahaemolyticus in seafood samples by loop-mediated isothermal amplification with hydroxynaphthol blue dye.

The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.

Loop-mediated isothermal amplification assay as a point-of-care diagnostic tool for Vibrio parahaemolyticus: recent developments and improvements.

INTRODUCTION: A number of DNA-based diagnostic tools have been developed for the detection of Vibrio parahaemolyticus in seafood. However, the loop-mediated isothermal amplification (LAMP) has distinct advantages with regards to its simplicity, speed and the ease of performing without any need for sophisticated equipment. Over the last decade, LAMP has emerged as a potential tool for the detection of V. parahaemolyticus. Area covered: The literature search was restricted to LAMP assay and its variants for the detection of V. parahaemolyticus. The focus in this review is to enlist the various techniques that have been developed using the principle of the LAMP towards improved simplicity, sensitivity and specificity of the assay. Expert commentary: LAMP assay and its variants are significantly faster and require minimum accessories compared to other DNA based molecular techniques such as PCR and their types. Despite the availability of several versions, LAMP-based diagnostics is not the first choice for the detection of V. parahaemolyticus in the seafood sector. Our recommendation would be to explore the possibilities of developing cost-effective LAMP kits and implementing these kits as point-of-care diagnostic tools for rapid and sensitive detection of pathogenic V. parahaemolyticus.

Comparative performance of TCBS and TSA for the enumeration of trh+ Vibrio parahaemolyticus by direct colony hybridization.

Vibrio parahaemolyticus is one of the important foodborne pathogens is of public health concern due to the emergence of pandemic strains causing disease outbreaks worldwide. We evaluated the DNA based colony hybridization technique for the detection and enumeration of total and pathogenic V. parahaemolyticus from the bivalve shellfish, clam using non-radioactive, enzyme-labeled probe targeting the tlh and trh genes, respectively. The digoxigenin (DIG) labeled probes designed in this study showed 100% specificity by dot blot assay. Colony hybridization using DIG probes was performed using both non-selective, trypticase soy agar (TSA) and the selective medium, thiosulfate citrate bile salts sucrose (TCBS) agar. Of 32 clam samples analyzed, 71.88% had>10,000 V. parahaemolyticus cells/g in TSA whereas it was 18.75% in case of TCBS. All the samples showed the presence of total V. parahaemolyticus in TSA and 97% in the case of TCBS. Interestingly, results of the trh+V. parahaemolyticus samples were quite high while using TCBS plates (62.5%) as compared to TSA (43.75%). However, the cell numbers obtained from TSA were higher than from TCBS. Several yellow colonies on TCBS turned out to be V. parahaemolyticus using colony hybridization, which was further confirmed by PCR and sucrose utilization test. Colony hybridization using DIG-labeled probe was found to be highly sensitive and could differentiate and enumerate pathogenic and non-pathogenic strains of V. parahaemolyticus. Since traditional methods are not only labor-intensive and time-consuming but also less sensitive, colony hybridization using DIG-labeled probes would be a useful alternative for the enumeration of V. parahaemolyticus in naturally contaminated seafood.