scholarly journals Specific Detection of the Wheat Blast Pathogen (Magnaporthe oryzae Triticum) by Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2018 ◽  
Vol 102 (12) ◽  
pp. 2550-2559 ◽  
Author(s):  
Jarred Yasuhara-Bell ◽  
Kerry F. Pedley ◽  
Mark Farman ◽  
Barbara Valent ◽  
James P. Stack
Keyword(s):  
Internal Control ◽  
Magnaporthe Oryzae ◽  
Nadh Dehydrogenase ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Wheat Blast ◽  
Negative Controls ◽  
Head Scab ◽  
Nad5 Gene

Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

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 Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

Loop-mediated isothermal amplification technique: principle, development and wide application in food safety

2020 ◽  
Vol 12 (46) ◽  
pp. 5551-5561
Author(s):  
Tianzeng Huang ◽  
Linzhi Li ◽  
Xing Liu ◽  
Qi Chen ◽  
Xueen Fang ◽  
...  
Keyword(s):  
Food Safety ◽  
Gene Amplification ◽  
Foodborne Pathogens ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Isothermal Conditions ◽  
Loop Mediated Isothermal Amplification ◽  
Novel Gene ◽  
Amplification Method ◽  
Amplification Technique

LAMP is a relatively novel gene amplification method under isothermal conditions with rapidity, and high specificity. It is widely applied in the field of food safety, such as in the detection of foodborne pathogens, GM, OP pesticides and so on

scholarly journals Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

2020 ◽  
Vol 21 (21) ◽  
pp. 7981
Author(s):  
Catalina Avendaño ◽  
Manuel Alfonso Patarroyo
Keyword(s):  
Neglected Tropical Diseases ◽  
Point Of Care ◽  
Low Cost ◽  
High Specificity ◽  
World Health Organisation ◽  
Isothermal Amplification ◽  
World Health ◽  
Parasitic Infections ◽  
River Blindness ◽  
Loop Mediated Isothermal Amplification

The World Health Organisation (WHO) has placed twenty diseases into a group known as neglected tropical diseases (NTDs), twelve of them being parasitic diseases: Chagas’ disease, cysticercosis/taeniasis, echinococcosis, food-borne trematodiasis, human African trypanosomiasis (sleeping sickness), leishmaniasis, lymphatic filariasis, onchocerciasis (river blindness), schistosomiasis, soil-transmitted helminthiasis (ascariasis, hookworm, trichuriasis), guinea-worm and scabies. Such diseases affect millions of people in developing countries where one of the main problems concerning the control of these diseases is diagnosis-based due to the most affected areas usually being far from laboratories having suitable infrastructure and/or being equipped with sophisticated equipment. Advances have been made during the last two decades regarding standardising and introducing techniques enabling diagnoses to be made in remote places, i.e., the loop-mediated isothermal amplification (LAMP) technique. This technique’s advantages include being able to perform it using simple equipment, diagnosis made directly in the field, low cost of each test and the technique’s high specificity. Using this technique could thus contribute toward neglected parasite infection (NPI) control and eradication programmes. This review describes the advances made to date regarding LAMP tests, as it has been found that even though several studies have been conducted concerning most NPI, information is scarce for others.

The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs

2013 ◽  
Vol 16 (1) ◽  
pp. 131-133 ◽  
Author(s):  
Ł. Adaszek ◽  
M. Jankowska ◽  
M. Kalinowski ◽  
T. Banach ◽  
D. Wułupek ◽  
...  
Keyword(s):  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Babesia Canis ◽  
Canine Babesiosis ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Amplification Assay ◽  
Analytical Laboratories ◽  
Babesia Canis Canis

Abstract The aim of this study was to use a rapid and easy DNA-based test, the loop-mediated isothermal amplification (LAMP), for diagnosis of Babesia canis canis infections in dogs. 10 DNA samples of 18S RNA-A and 10 DNA samples of 18S RNA-B of B. canis canis were used in the study. LAMP method could successfully detect DNA in all examined samples down to 0.1 pg dilution. Obtained results suggest that this method has high specificity and sensitivity and can be applied in analytical laboratories in diagnosis of canine babesiosis.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) for Detection of Various Organisms: A Review

2017 ◽  
Vol 11 (2) ◽  
pp. 20-27
Author(s):  
Arifa Akram
Keyword(s):  
Nucleic Acid ◽  
High Specificity ◽  
Disease Diagnosis ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Isolation And Identification ◽  
Strand Displacement Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Prophylactic Measures

Disease diagnosis is important for implementation of proper therapeutic and prophylactic measures. Traditionally, disease diagnosis was depends upon isolation and identification of the causative organisms. This was followed by serology and after that molecular method. Molecular tests are valuable when early diagnosis is important. For this purpose, nucleic acid amplification (PCR, nucleic acid sequence-based amplification, self-sustained sequence replication, strand displacement amplification) is one of the most valuable tools not only for the diagnosis of infectious diseases but also used in advanced level research. The Loop-Mediated Isothermal Amplification (LAMP) is a unique nucleic acid amplification technique for diagnosis of various pathogens introduced at 2000 by Notomi and his colleagues which is simple, easy, rapid and cost effective when compared to PCR due to its high specificity, sensitivity, and rapidity. It uses a set of six primers and a DNA polymerase with stranddisplacement activity. Major advantage of LAMP method is its cost-effectiveness as it can be done simply by using waterbath or heating block. Bangladesh J Med Microbiol 2017; 11 (2): 20-27

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata

2017 ◽  
Vol 100 (1) ◽  
pp. 99-103 ◽  
Author(s):  
Xinyue Zhang ◽  
Guojie Xu ◽  
Huaqi Tang ◽  
Yanpeng Li ◽  
Chunsheng Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Alternaria Alternata ◽  
Lamp Assay ◽  
High Specificity ◽  
Etiologic Agent ◽  
Isothermal Amplification ◽  
Pcr Assay ◽  
The Real ◽  
Loop Mediated Isothermal Amplification

Abstract Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma,and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/μL of A. alternata genomic DNA with high specificity. In addition, both the LAMP assay and the real-time PCR assay can be used for quantification of A. alternata. Although the newly developed LAMP assay is more rapid and specific in A. alternata identification, the real-time PCR assay is more precise in quantitation analysis.

scholarly journals Evaluation of the real-time fluorescence loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Xu-Guang Guo ◽  
Ya-Ru Zhuang ◽  
Jin-Zhou Wen ◽  
Tian-Ao Xie ◽  
Ye-Ling Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Agalactiae ◽  
Rapid Detection ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Bacterial Strains ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Strand Displacement ◽  
Time Amplification

Abstract Streptococcus agalactiae is a major pathogenic bacterium causing perinatal infections in humans. In the present study, a novel real-time fluorescence loop-mediated isothermal amplification technology was successfully developed and evaluated for the detection of S. agalactiae in a single reaction. Six specific primers were designed to amplify the corresponding six regions of fbs B gene of S. agalactiae, using Bst DNA polymerase with DNA strand displacement activity at a constant temperature for 60 min. The presence of S. agalactiae was indicated by the fluorescence in real-time. Amplification of the targeted gene fragment was optimized with the primer 1 in the current setup. Positive result was only obtained for Sa by Real-LAMP among 10 tested relevant bacterial strains, with the detection sensitivity of 300 pg/µl. Real-LAMP was demonstrated to be a simple and rapid detection tool for S. agalactiae with high specificity and stability, which ensures its wide application and broad prospective utilization in clinical practice for the rapid detection of S. agalactiae.

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