scholarly journals Qualitative and Quantitative Detection of Potentially Virulent Vibrio parahaemolyticus in Drinking Water and Commonly Consumed Aquatic Products by Loop-Mediated Isothermal Amplification

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 10
Author(s):  
Zhengke Shen ◽  
Yue Liu ◽  
Lanming Chen
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Limit Of Detection ◽  
Uv Light ◽  
Infection Process ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Qualitative And Quantitative ◽  
Loop Mediated Isothermal Amplification

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infection, and septicemia in humans. In this study, a simple, specific, and user-friendly diagnostic tool was developed for the first time for the qualitative and quantitative detection of toxins and infection process-associated genes opaR, vpadF, tlh, and ureC in V. parahaemolyticus using the loop-mediated isothermal amplification (LAMP) technique. Three pairs of specific inner, outer, and loop primers were designed for targeting each of these genes, and the results showed no cross-reaction with the other common Vibrios and non-Vibrios pathogenic bacteria. Positive results in the one-step LAMP reaction (at 65 °C for 45 min) were identified by a change to light green and the emission of bright green fluorescence under visible light and UV light (302 nm), respectively. The lowest limit of detection (LOD) for the target genes ranged from 1.46 × 10−5 to 1.85 × 10−3 ng/reaction (25 µL) for the genomic DNA, and from 1.03 × 10−2 to 1.73 × 100 CFU/reaction (25 µL) for the cell culture of V. parahaemolyticus. The usefulness of the developed method was demonstrated by the fact that the bacterium could be detected in water from various sources and commonly consumed aquatic product samples. The presence of opaR and tlh genes in the Parabramis pekinensis intestine indicated a risk of potentially virulent V. parahaemolyticus in the fish.

scholarly journals Loop Mediated Isothermal Amplification as a Rapid Molecular Method for Pseudomonas aeruginosa T3SS ExoY Gene Septicemia Detection in Beta- Lactamase Species Co-Infections

2021 ◽  
pp. 19-29
Author(s):  
J. Mageto Ombega ◽  
Zhao-Hua Zhong
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Limit Of Detection ◽  
Uv Light ◽  
Lamp Assay ◽  
Chronically Ill ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Molecular Technique ◽  
Loop Mediated Isothermal Amplification

Background: Pseudomonas aeruginosa is among the most important causative agent of infection in chronically ill patients admitted in hospitals globally. Coupled with its, mixed symptomatology, rapid drug resistance tendency and its causation of severe disease, a fast, reliable and affordable diagnostic technique is required to enable healthcare providers expeditiously mitigate its progression and eventual treatment. The Loop-Mediated Isothermal Amplification (LAMP) technique has the potential to serve as a simple, rapid, specific, sensitive and cost-effective point-of-care diagnostic tool. Broad Objective: To investigate Loop Mediated Isothermal Amplification as a molecular technique for microbial diagnostic and prognostic predictor.   Study Design: This study was aimed at evaluating LAMP assay against Simple Polymerase chain reaction and Multiplex PCR on the diagnosis of P. aeruginosa in mixed clinical samples. Materials and Methods: This study developed P. aeruginosa Loop Mediated Isothermal Amplification (PaLAMP) assay to target the ExoY gene with appropriate primer testing and validation procedures. Culture of patient bacterial samples was done on MHA and MHB medium, grown overnight in an Incubator and a incubating shaker at 37oc respectively. Housekeeping gene were identified through online bioinformatics and blasted against known sequences. A set of 6 primers, comprising 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP), and 2 loop primers (FLP and BLP), were designed. Microbial DNA extraction was done followed by PCR amplification as a classical identification using LAMP outer primers 9(F3 and B3). LAMP amplicons were detected by real time turbidimetry (LA-500) at 64°C for 40 minutes as well as under UV light with 1.0 μl of 1/10-diluted original SYBR Green I. Results: LAMP validation against traditional PCR shows a high limit of detection at 10-6ng/µl compared to 10-5ng/µl for PCR. The findings are consistent with outcomes for real time turbidimetric outcomes. Real time LAMP turbidimetric results was cross validated by direct observation through SYBR fluorescence under UV light for positive P. aeruginosa detection through positive amplification. Conclusion: Thus far, Loop mediated isothermal amplification show significantly high limit of detection comparable to standard PCR, its use in field based diagnosis offers an opportunity for a cheap, reliable and faster method to determine disease trends and therapy approaches. This method can be applied in primary care to enhance accuracy in diagnosis and thereby prompt initiation of mitigation treatment regimens.

scholarly journals Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification

2021 ◽  
Author(s):  
DAILING CHEN ◽  
ZHILI LIANG ◽  
SHUNLIN REN ◽  
WALID ALALI ◽  
Lanming Chen
Keyword(s):  
Vibrio Cholerae ◽  
Target Genes ◽  
Limit Of Detection ◽  
Molecular Probes ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Aquatic Products ◽  
Standard Polymerase Chain Reaction ◽  
Visualized Detection

Vibrio cholerae  can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was for the first time developed based on loop-mediated isothermal amplification (LAMP) for detection of very important virulence-related genes ace , zot , cri , and  nanH for toxins and infection process of V. cholerae . Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65 o C for 40 min. Positive results were simply inspected by the production of light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection (LOD) of the LAMP method ranged from 1.85-2.06 pg/reaction of genomic DNA or 2.50-4.00×10 2  CFU/reaction for target genes of cell culture of V. cholerae , which was more sensitive than standard polymerase chain reaction (PCR). Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by the absence from all water samples from various sources. However, high occurrences of nanH  gene were observed in intestine samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae  in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.

scholarly journals A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

2021 ◽  
Vol 18 (9) ◽  
pp. 4574
Author(s):  
Kai Chen ◽  
Biao Ma ◽  
Jiali Li ◽  
Erjing Chen ◽  
Ying Xu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Quantitative Detection ◽  
Bacterial Strains ◽  
Food Borne Pathogens ◽  
Food Borne

Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA–RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA–RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9–114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA–RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals Dual-Modal Assay Kit for the Qualitative and Quantitative Determination of the Total Water Hardness Using a Permanent Marker Fabricated Microfluidic Paper-Based Analytical Device

Chemosensors ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 97
Author(s):  
Oyejide Damilola Oyewunmi ◽  
Seyed Hamid Safiabadi-Tali ◽  
Sana Jahanshahi-Anbuhi
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Limit Of Detection ◽  
Low Cost ◽  
Water Hardness ◽  
Sample Introduction ◽  
World Health ◽  
Total Hardness ◽  
Quantitative Detection ◽  
Qualitative And Quantitative ◽  
Analytical Device

A dip-and-read microfluidic paper-based analytical device (µPAD) was developed for the qualitative and quantitative detection of the total hardness of water. To create well-defined hydrophobic barriers on filter paper, a regular office printer and a commercially available permanent marker pen were utilized as a quick and simple technique with easily accessible equipment/materials to fabricate µPAD in new or resource-limited laboratories without sophisticated equipment. After a wettability and barrier efficiency analysis on the permanent marker colors, the blue and green ink markers exhibited favorable hydrophobic properties and were utilized in the fabrication of the developed test devices. The device had five reaction and detection zones modeled after the classification given by the World Health Organization (WHO), so qualitatively it determined whether the water was ‘soft’, ‘moderately hard’, ‘hard’, or ‘very hard’ by changing color from blue to pink in about 3 min. The device was also used to introduce an alternative colorimetric reaction for quantitative analysis of the water hardness without the need for ethylenediaminetetraacetic acid (EDTA) and without compromising the simplicity and low cost of the device. The developed µPAD showed a calculated limit of detection (LOD) of 0.02 mM, which is at least 80% less than those of commercially available test strips and other reported µPADs, and the results of the real-world samples were consistent with those of the standard titration (with EDTA). In addition, the device exhibited stability for 2 months at room and frigid condition (4 °C) and at varying harsh temperatures from 25 to 100 °C. The results demonstrate that the developed paper-based device can be used for rapid, on-site analysis of water with no interferences and no need for a pipette for sample introduction during testing.

scholarly journals Rapid detection of cytochrome cd1-containing nitrite reductase encoding gene nirS of denitrifying bacteria with loop-mediated isothermal amplification assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xuzhi Zhang ◽  
Qianqian Yang ◽  
Qingli Zhang ◽  
Xiaoyu Jiang ◽  
Xiaochun Wang ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Denitrifying Bacteria ◽  
Isothermal Amplification ◽  
Target Sequence ◽  
Bacterial Cells ◽  
Loop Mediated Isothermal Amplification ◽  
Order Of Magnitude

Abstract The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

scholarly journals q-LAMP Assays for the Detection of Botryosphaeria dothidea Causing Chinese Hickory Canker in Trunk, Water, and Air Samples

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3142-3149
Author(s):  
Q. W. Wang ◽  
C. Q. Zhang
Keyword(s):  
Limit Of Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Botryosphaeria Dothidea ◽  
Canker Disease ◽  
Air Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Optimum Reaction ◽  
Epidemiological Characteristics ◽  
Trunk Canker

Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea, which could be applied widely in epidemiology, forecast, and management of tree canker disease.

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