scholarly journals Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0250808
Author(s):  
Satyaki Ghosh ◽  
David L. Straus ◽  
Christopher Good ◽  
Vipaporn Phuntumart
Keyword(s):  
Quantitative Pcr ◽  
Its Region ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Lamp Method ◽  
Specific Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Freshwater Aquaculture ◽  
Qpcr Method ◽  
Important Disease

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.

scholarly journals Development and comparison of Loop-Mediated Isothermal Amplification with Quantitative PCR for the specific detection of Saprolegnia spp.

2021 ◽  
Author(s):  
Satyaki Ghosh ◽  
David L Straus ◽  
Christopher Good ◽  
Vipaporn Phuntumart
Keyword(s):  
Quantitative Pcr ◽  
Its Region ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Lamp Method ◽  
Specific Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Freshwater Aquaculture ◽  
Qpcr Method ◽  
Important Disease

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia.  Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20-60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.

Species-specific detection of medically important aspergilli by a loop-mediated isothermal amplification method in chronic pulmonary aspergillosis

2019 ◽  
Vol 57 (6) ◽  
pp. 703-709
Author(s):  
Kazuya Tone ◽  
Junko Suzuki ◽  
Mohamed Mahdi Alshahni ◽  
Kazuyoshi Kuwano ◽  
Koichi Makimura
Keyword(s):  
Sensitivity And Specificity ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Detection ◽  
Pulmonary Aspergillosis ◽  
Loop Mediated Isothermal Amplification ◽  
Group A ◽  
Chronic Pulmonary Aspergillosis ◽  
Species Specific

AbstractChronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid Identification of Dendrobium officinale

2018 ◽  
Author(s):  
Lu Yang ◽  
Hua Zhou ◽  
Huili Lai ◽  
Fei Fu ◽  
Wenru Wu
Keyword(s):  
Color Change ◽  
Dna Barcode ◽  
Its Region ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Dendrobium Officinale ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Species Specific

Background: Dendrobium officinale is not only an ornamental plant, but also a valuable medicinal herb that is both effective and widely used in traditional Chinese medicine. However, distinguishing D. officinale from other Dendrobium species is usually a difficult task that need much time and complex technologies due to their very similar external morphologies. The aim of this study is to develop a fast, even on-spot approach to identify D. officinale. Methods: We used DNA barcode-based loop-mediated isothermal amplification (LAMP) method with species-specific LAMP primers targeting the internal transcribed spacer (ITS) region of the rDNA of D. officinale. LAMP reaction time and temperature were optimized and the specificity and sensitivity of LAMP species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of D. officinale and allowed for rapid amplification (within 40 min) of the ITS region under a constant and mild temperature range of 65 °C without using thermocyclers. Besides, by using SYBR® Green I dye as the color developing agent, the color change was easily observed with naked eye. Reaction mixture containing DNA of D. officinale changed from orange to green, while the other Dendrobium species and the negative control retained original orange color. The specificity of this LAMP-based method was confirmed by testing 17 samples of D. officinale and 32 adulterant samples from other Dendrobium species. Conclusions: This LAMP-based rapid identification method does not require expensive equipment or specialized techniques and can be used in field surveys for accurate and fast on site identification.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

Sign in / Sign up

Export Citation Format

Share Document