scholarly journals LAMP Assay for the Detection of Echinococcus multilocularis Eggs Isolated from Canine Faeces by a Cost-Effective NaOH-Based DNA Extraction Method

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 847
Author(s):  
Barbara J. Bucher ◽  
Gillian Muchaamba ◽  
Tim Kamber ◽  
Philipp A. Kronenberg ◽  
Kubanychbek K. Abdykerimov ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Extraction ◽  
Sensitivity And Specificity ◽  
Echinococcus Multilocularis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Detection Methods ◽  
Simple Method ◽  
Commercial Kits

The detection of Echinococcus multilocularis in infected canids and the environment is pivotal for a better understanding of the epidemiology of alveolar echinococcosis in endemic areas. Necropsy/sedimentation and counting technique remain the gold standard for the detection of canid infection. PCR-based detection methods have shown high sensitivity and specificity, but they have been hardly used in large scale prevalence studies. Loop-mediated isothermal amplification (LAMP) is a fast and simple method to detect DNA with a high sensitivity and specificity, having the potential for field-application. A specific LAMP assay for the detection of E. multilocularis was developed targeting the mitochondrial nad1 gene. A crucial step for amplification-based detection methods is DNA extraction, usually achieved utilising silica-gel membrane spin columns from commercial kits which are expensive. We propose two cost-effective and straightforward methods for DNA extraction, using NaOH (method 1A) and InstaGeneTM Matrix (method 1B), from isolated eggs circumventing the need for commercial kits. The sensitivity of both assays with fox samples was similar (72.7%) with multiplex-PCR using protocol 1A and LAMP using protocol 1B. Sensitivity increased up to 100% when testing faeces from 12 foxes infected with more than 100 intestinal stages of E. multilocularis. For dogs, sensitivity was similar (95.4%) for LAMP and multiplex-PCR using protocol 1B and for both methods when DNA was extracted using protocol 1A (90.9%). The DNA extraction methods used here are fast, cheap, and do not require a DNA purification step, making them suitable for field studies in low-income countries for the prevalence study of E. multilocularis.

scholarly journals Marine Toxins Detection by Biosensors Based on Aptamers

Toxins ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 1 ◽  
Author(s):  
Wei Ye ◽  
Taomei Liu ◽  
Weimin Zhang ◽  
Muzi Zhu ◽  
Zhaoming Liu ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
High Efficiency ◽  
Rapid Development ◽  
High Sensitivity ◽  
Detection Methods ◽  
Marine Toxins ◽  
Time Saving ◽  
Low Detection Limit ◽  
The Future

Marine toxins cause great harm to human health through seafood, therefore, it is urgent to exploit new marine toxins detection methods with the merits of high sensitivity and specificity, low detection limit, convenience, and high efficiency. Aptasensors have emerged to replace classical detection methods for marine toxins detection. The rapid development of molecular biological approaches, sequencing technology, material science, electronics and chemical science boost the preparation and application of aptasensors. Taken together, the aptamer-based biosensors would be the best candidate for detection of the marine toxins with the merits of high sensitivity and specificity, convenience, time-saving, relatively low cost, extremely low detection limit, and high throughput, which have reduced the detection limit of marine toxins from nM to fM. This article reviews the detection of marine toxins by aptamer-based biosensors, as well as the selection approach for the systematic evolution of ligands by exponential enrichment (SELEX), the aptamer sequences. Moreover, the newest aptasensors and the future prospective are also discussed, which would provide thereotical basis for the future development of marine toxins detection by aptasensors.

scholarly journals Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2381
Author(s):  
Katarzyna Skrzypek ◽  
Jacek Karamon ◽  
Małgorzata Samorek-Pieróg ◽  
Joanna Dąbrowska ◽  
Maciej Kochanowski ◽  
...  
Keyword(s):  
Positive Result ◽  
Multiplex Pcr ◽  
Dna Extraction ◽  
Echinococcus Multilocularis ◽  
Nested Pcr ◽  
Genetic Material ◽  
Extraction Methods ◽  
Red Foxes ◽  
Stool Samples ◽  
Positive Results

(1) Background: Due to the increasing distribution of Echinococcus multilocularis infections in final hosts, epidemiological investigations are important for recognizing the spreading pattern of this parasite and also to estimate risk infection for humans. (2) Methods: Investigations were conducted with two commercial kits dedicated for DNA extraction from feces: ZR Fecal DNA Mini Prep (Zymo Research, Freiburg, Germany) and QIAamp FAST DNA Stool Mini Kit (Qiagen, Hilden, Germany) (marked as Z and Q), together with two common PCR protocols (nested PCR and multiplex PCR). The goal was to compare their efficiency in detecting the genetic material of E. multilocularis in the samples of feces. Stool samples from red foxes were collected in a highly endemic area in Poland. Sedimentation and counting technique (SCT) was used as a reference method. (3) Results: From 48 samples, 35 were positive in SCT. Further investigations showed that 40.0% of samples (from those with SCT positive result) after Z-DNA extraction and 45.7% after Q-DNA extraction gave positive results in nested PCR. In multiplex PCR, positive results were obtained in 54.3% of samples after Z isolation and 48.6% of samples after Q. Additionally, one sample that resulted in being negative in SCT gave a positive result in PCR. The number of worms detected in the intestines had no influence on PCR results. (4) Conclusions: Both of the extraction methods showed similar efficiency in DNA isolation and dealing with inhibitors; however, they showed relatively low sensitivity. This was probably caused by degradation of genetic material in the field-collected samples.

HIGHLY SENSITIVE H7N9 AVIAN INFLUENZA VIRUS DETECTION USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION AND OLIGONUCLEOTIDE MICROARRAY

2015 ◽  
Vol 41 (04) ◽  
pp. 251-255
Author(s):  
Lih-Chiann Wang ◽  
Dean Huang
Keyword(s):  
Avian Influenza ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Oligonucleotide Microarray ◽  
Influenza Viruses ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Viral Copy Number

H7N9 avian influenza viruses have circulated in the human population and poultry flocks in China since 2013. H7N9 virus monitoring is imperative in Taiwan due to the frequent contact between China and Taiwan. Traditional viral molecular detection methods using RT-PCR and sequencing are time- and labor-intensive, thus a simpler and cheaper method with high sensitivity and specificity is worth developing. We successfully detected human and wild bird H7N9 viruses in this study using reverse transcription loop-mediated isothermal amplification (RT-LAMP) and oligonucleotide microarray. The detection limit was as low as one viral copy number. The specific matching reaction between the templates and microarray probes during hybridization ensured the high detection effectiveness. The excellent sensitivity and specificity of the RT-LAMP-microarray makes it a powerful H7N9 surveillance approach in Taiwan.

scholarly journals Using a Loop-Mediated Isothermal Amplification (LAMP) Assay and Direct DNA Extraction Method from Wood for Rapid Detection of Verticillium dahliae in Olive Trees

2017 ◽  
Vol 14 (2) ◽  
pp. 727-734
Author(s):  
Saba Aslani ◽  
Ghasemali Garoosi ◽  
Hossein Jafary
Keyword(s):  
Dna Extraction ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Lamp Assay ◽  
Cost Effective ◽  
Pathogenic Fungi ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Minimum Requirements ◽  
Olive Trees

ABSTRACT: Verticillium wilt, which is caused by the fungus Verticillium dahliae, is one of the most important olive diseases worldwide. There are many ways to extract DNA from plant pathogenic fungi and from plant tissues for molecular-based diagnostic assays. LAMP is a new and sensitive molecular-based technique used for detection of plant pathogenic agents with minimum requirements needed. In this study, we tried to achieve a simple, cost effective and efficient method of DNA extraction from both Verticillium dahliae fungus and from infected wood samples in order to run a loop-mediated isothermal amplification (LAMP) assay. Efficiency of three DNA isolation methods from both mycelia and infected wood samples was evaluated. For this purpose, wood samples from infected olive trees were collected from Tarom region in Zanjan province and the samples were cultured on the media. The fungus was isolated and identified as V. dahliae based on morphological features. Then the genomic DNA was extracted using traditional CTAB method, fast NaOH method and direct isolation method from infected wood samples. After assessment of the quality and the quantity of the extracted DNA samples, a LAMP assay was ran using specific primer pairs and the DNA templates extracted using three different methods. In spite of the significant differences in the quantity of DNA samples, LAMP assay could successfully detect the fungus in all samples. The improved direct isolation of the DNA of V. dahlia from infected wood, followed by a LAMP assay could considerably shortened the detection process of the fungus and hence is a suitable method for screening of olive trees and saplings against Verticillium wilt disease.

scholarly journals Combination of 16S rRNA and procalcitonin in diagnosis of neonatal clinically suspected sepsis

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051989241
Author(s):  
Renqiang Yu ◽  
Qin Zhou ◽  
Shanyu Jiang ◽  
Yingzi Mei ◽  
Min Wang
Keyword(s):  
16S Rrna ◽  
Sensitivity And Specificity ◽  
Bacterial Culture ◽  
High Sensitivity ◽  
16S Rrna Sequencing ◽  
Detection Methods ◽  
Suspected Sepsis ◽  
Rrna Sequencing ◽  
Positive Rate ◽  
Sensitivity Specificity

Objective To investigate the application of 16S rRNA in diagnosing patients with neonatal sepsis. Methods We studied 60 consecutive neonatal patients with clinically suspected sepsis and 20 non-infective cases as controls. All patients were diagnosed with sepsis by clinical and experimental criteria. Clinical characteristics were recorded and 16S rRNA sequencing was conducted for all patients. The sensitivity, specificity, and accuracy of the detection methods were analyzed. Results The detection limit of 16S rRNA sequencing was 1 × 102 CFU/mL. For suspected sepsis, the positive rate of 16S rRNA detection was 93.3%, which was similar to that of procalcitonin detection (85%), and was significantly higher than that of bacterial culture (51.7%). The specificity of procalcitonin detection (74.1%) was significantly lower than that of 16S rRNA detection (100%). Moreover, the combination of 16S rRNA and procalcitonin detection showed a sensitivity of 100%, specificity of 74.1%, and accuracy of 92.0%. For proven sepsis, the sensitivity and specificity of 16S rRNA detection were both 100.0%, and those for procalcitonin were 87.1% and 87.0%, respectively. Conclusion Detection of 16S rRNA has high sensitivity and specificity in diagnosing sepsis. The combination of 16S rRNA and procalcitonin has even better sensitivity with acceptable specificity.

scholarly journals Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (12) ◽  
pp. 4008-4016 ◽  
Author(s):  
Siyi Chen ◽  
Fei Wang ◽  
John C. Beaulieu ◽  
Rebecca E. Stein ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Sample Treatment ◽  
Propidium Monoazide ◽  
Simple Method ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTRecent outbreaks linked toSalmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targetingSalmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killedSalmonellacells with concentrations up to 108CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viableSalmonellacells in pure culture and 6.1 × 103to 6.1 × 104CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viableSalmonellacell numbers was high (R2= 0.949 to 0.993), with a quantification range (102to 105CFU/reaction in pure culture and 104to 107CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viableSalmonellacells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce.

scholarly journals Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of ‘Candidatus Liberibacter asiaticus’

2018 ◽  
Vol 108 (2) ◽  
pp. 292-298 ◽  
Author(s):  
Binghai Lou ◽  
Yaqin Song ◽  
Moytri RoyChowdhury ◽  
Chongling Deng ◽  
Ying Niu ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
High Sensitivity ◽  
Cost Effective ◽  
Detection Methods ◽  
Displacement Reaction ◽  
Candidatus Liberibacter Asiaticus ◽  
Highly Sensitive ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of ‘Candidatus Liberibacter asiaticus’, a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of ‘Ca. L. asiaticus’ with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of ‘Ca. L. asiaticus’.

scholarly journals Role of Phytopathogenic Fungi in Forest Plant Breeding–Development of DNA-Based Quick Tests for Quality Assurance in Forest Plant Production

2020 ◽  
Vol 3 (1) ◽  
pp. 96
Author(s):  
Kristin Morgenstern ◽  
Jens-Ulrich Polster ◽  
Birgit Reiche ◽  
Patrick Schützel ◽  
Imke Hutter ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Constant Temperature ◽  
Plant Pathogens ◽  
Lamp Assay ◽  
Its Region ◽  
Cost Effective ◽  
Conventional Polymerase Chain Reaction ◽  
Lamp Reaction ◽  
Plant Production ◽  
Technical Effort

The production of healthy seed and plant material is a fundamental prerequisite for the establishment of ecologically stable and economically productive forest stands. As in the past, forest plant production is nevertheless threatened by harmful biotic factors, with new, invasive species playing an increasingly significant role as a result of climate change and globalization. DNA-based methods have significantly accelerated the detection of plant pathogens, but are still time-consuming, costly, and require extensive equipment. Loop-mediated isothermal amplification (LAMP) is an efficient and cost-effective alternative to conventional polymerase chain reaction (PCR). The LAMP reaction is performed as a one-step assay at constant temperature and can be evaluated visually. The ongoing project “TreeLAMP” has led to the establishment of an LAMP assay for Rhabdocline pseudotsugae, one of the most important needle pathogens of Douglas fir. To date, 32 sets of LAMP primers have been derived from the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and tested. The results show clear differences between the primer sets both in terms of reaction rate and concentration of amplified products. Following extensive work targeting the optimization of the LAMP reaction, a method is now available that enables the reliable detection of R. pseudotsugae at a constant temperature (65 °C) and after a reaction time of 1.5 h. The detection limit is currently 0.02 pg/µL of R. pseudotsugae DNA. The current focus of the project is the optimization of DNA extraction. In addition to conventional DNA kits, methods that are specially adapted to the detection procedure are also under investigation. These will allow DNA extraction to occur faster and without any great technical effort.

scholarly journals A Simple Colorimetric Molecular Detection of Novel Coronavirus (COVID-19), an Essential Diagnostic Tool for Pandemic Screening

2020 ◽  
Author(s):  
Pazhanimuthu Annamalai ◽  
Madhu Kanta ◽  
Pazhanivel Ramu ◽  
Baskar Ravi ◽  
Kokilavani Veerapandian ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Recent Outbreak ◽  
Pcr Method ◽  
Novel Coronavirus ◽  
Virus Isolates

AbstractThe recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.

scholarly journals Evaluation of RNA extraction free method for detection of SARS-COV-2 in salivary samples for mass screening for COVID-19

2021 ◽  
Author(s):  
Sally A. Mahmoud ◽  
Esra Ibrahim ◽  
Subhashini Ganesan ◽  
Bhagyashree Thakre ◽  
Juliet G Teddy ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Gold Standard ◽  
Rna Extraction ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Kappa Coefficient ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Good Percentage

AbstractIn this current COVID - 19 pandemic, there is a dire need for cost effective and less time-consuming alternatives for SARS-COV-2 testing. The RNA extraction free method for detecting SARS-COV-2 in saliva is a promising option, this study found that it has high sensitivity (85.34%), specificity (95.04%) and was comparable to the gold standard nasopharyngeal swab. The method showed good percentage of agreement (kappa coefficient) 0.797 between salivary and NPS samples. However, there are variations in the sensitivity and specificity based on the RT-PCR kit used. The Thermo Fischer-Applied biosystems showed high sensitivity, PPV and NPV but also showed higher percentage of invalid reports. Whereas the BGI kit showed high specificity, better agreement (kappa coefficient) between the results of saliva and NPS samples and higher correlation between the Ct values of saliva and NPS samples. Thus, the RNA extraction free method for salivary sample serves as an effective alternative for SARS-CoV 2-testing.

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