scholarly journals Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico

2021 ◽  
Vol 47 (4) ◽  
Author(s):  
Ismael Mendoza-Avilés ◽  
Carla A Muñoz-Rojas ◽  
Mario Rojas ◽  
Norma Estrada
Keyword(s):  
Disease Transmission ◽  
Target Genes ◽  
Reaction Times ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
White Shrimp ◽  
Loop Mediated Isothermal Amplification ◽  
Aquaculture Industry ◽  
Candidate Target ◽  
Primer Sets

Loop-mediated isothermal amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification under isothermal conditions, and it has served as a powerful diagnostic tool. In this study, we used LAMP to develop a diagnostic protocol for detecting Vibrio parahaemolyticus and white spot syndrome virus in whiteleg shrimp, and Perkinsus spp. in Crassostrea spp. in Mexico. These pathogens are associated with different diseases and are considered a threat in the aquaculture industry. Infected and uninfected oysters and shrimp were obtained from farms in the northwest coast of Mexico to standardize the LAMP assay. We determined the candidate target genes in the first-round analysis of many sets of primers, and then we chose a set of primers that successfully amplified with Mexican samples. We optimized the LAMP reactions for each pathogen with the chosen primer sets using temperature gradients from 61 to 65 ºC, DNA concentrations from 2.5 pg to 250.0 ng, and reaction times from 10 to 60 min. This study established a diagnostic procedure for detecting pathogens in oysters and shrimp from Mexico. Early diagnosis and treatment of pathogens can immensely reduce disease transmission in aquaculture farms.

scholarly journals Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2016 ◽  
Vol 100 (9) ◽  
pp. 1804-1812 ◽  
Author(s):  
X. Zhang ◽  
T. C. Harrington ◽  
J. C. Batzer ◽  
R. Kubota ◽  
N. A. Peres ◽  
...  
Keyword(s):  
Pure Culture ◽  
Detection Threshold ◽  
Lamp Assay ◽  
Its Region ◽  
Isothermal Amplification ◽  
Fruit Rot ◽  
Colletotrichum Acutatum ◽  
Initial Inoculum ◽  
Loop Mediated Isothermal Amplification ◽  
Primer Sets

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the β-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml−1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml−1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml−1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

scholarly journals Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248042
Author(s):  
Woong Sik Jang ◽  
Da Hye Lim ◽  
Jung Yoon ◽  
Ahran Kim ◽  
Minsup Lim ◽  
...  
Keyword(s):  
South Korea ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Limits ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Primer Sets

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

Rapid detection of peach shoot blight caused by Phomopsis amygdali utilizing a new target gene identified from genome sequences within loop-mediated isothermal amplification

Plant Disease ◽  
2021 ◽  
Author(s):  
Lina Yang ◽  
Liang Zhang ◽  
Jun Cao ◽  
Lingyun Wang ◽  
Hengsong Shi ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Southern China ◽  
Reaction System ◽  
Lamp Assay ◽  
Pcr Primers ◽  
Isothermal Amplification ◽  
Detection Rates ◽  
Loop Mediated Isothermal Amplification ◽  
Shoot Blight ◽  
Primer Sets

Peach shoot blight (PSB) caused by Phomopsis amygdali is a serious threat to the healthy development of the peach industry and leads to 30-50 % damage to peach production in southern China. In this study, loop-mediated isothermal amplification (LAMP) technology was used to detect the P. amygdali target of a gene of GME6801 that was unique in the whole genome of the pathogen compared with that of Diaporthe (Phomopsis) longicolla TWH P74, Fusurium graminearum PH-1, Colletotrichum gloeosporioides SMCG1 and Magnaporthe oryzae 70-15. Blast comparison of this gene sequence in NCBI database showed that no homologous sequences were found. Therefore, the gene sequence of GME6801 was used to design two pairs of LAMP primers and one pair of PCR primers. The results showed that both of primer sets were specific to the 15 strains of P. amygdali, and the other 15 fungal strains presented negative reactions, similar to the control. In addition, 50 pg of genomic DNA of P. amygdali in a 25 μl reaction system could be detected by LAMP assay which was 100 times more sensitive than PCR. Furthermore, the GME6801 LAMP assay was used to detect artificially inoculated twigs of the pathogen, disease twigs within significantly symptomatic PSB in the field and healthy twigs in the same orchard, with the detection rates of 100%, 75% and 20.8%, respectively. However, the detection rates of conventional PCR were separately 100%, 62.5% and 16.7%. The results indicated that GME6801-based LAMP could be used for P. amygdali detection as its specificity, sensitivity and simplicity. This study provides a rapid experimental basis for the identification and prediction of P. amygdali that causes PSB and is beneficial for precise prevention and control of the disease.

scholarly journals Molecular Detection of QoI Resistance in Colletotrichum gloeosporioides Causing Strawberry Anthracnose Based on Loop-Mediated Isothermal Amplification Assay

Plant Disease ◽  
2019 ◽  
Vol 103 (6) ◽  
pp. 1319-1325 ◽  
Author(s):  
J. Y. Wu ◽  
X. R. Hu ◽  
C. Q. Zhang
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Lamp Assay ◽  
Practical Method ◽  
Conventional Polymerase Chain Reaction ◽  
Isothermal Amplification ◽  
Time Saving ◽  
Loop Mediated Isothermal Amplification ◽  
Quinone Outside Inhibitor ◽  
Strawberry Anthracnose ◽  
Primer Sets

Anthracnose is one of the most common diseases in strawberry plants. Colletotrichum gloeosporioides is the major cause of anthracnose in China, including Zhejiang Province. Early, specific, reliable, and time-saving detection is urgently needed to prevent the further spread of C. gloeosporioides, guiding farmers to utilize chemicals to control anthracnose. In this study, we showed that the high resistance to pyraclostrobin, caused by a point mutation at codon 143 (GGT→GCT) in the cytochrome b gene of C. gloeosporioides was prevalent in the strawberry growing regions, and we developed a loop-mediated isothermal amplification (LAMP) assay as a detection method. Primer sets S0 and S4 could be used to specifically detect C. gloeosporioides isolates and the G143A mutations, respectively. A detection limit of 10−2 ng (10 pg), which is at least 10-fold more sensitive than conventional polymerase chain reaction, was achieved by the LAMP assay. Here, we utilized lateral-flow devices (LFDs), nitrocellulose membranes that can absorb nucleic acids, to acquire the total genomic DNA of strawberry plants within 2 min. The LFD membranes were used as DNA templates for the LAMP assays to accurately detect strawberry plants infected with C. gloeosporioides. This diagnostic method for strawberry anthracnose was accomplished within 1 h, including the sample preparation and LAMP assays. Collectively, we developed a sensitive and practical method for monitoring C. gloeosporioides and its quinone outside inhibitor–resistant mutants. The LAMP assay for detection of C. gloeosporioides in strawberry plants has great potential for rapid strawberry anthracnose surveillance and will provide farmers with advice on preventing C gloeosporioides at the early stages of strawberry development.

scholarly journals A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac

2019 ◽  
Vol 20 (8) ◽  
pp. 1937 ◽  
Author(s):  
Miaomiao Sun ◽  
Hao Liu ◽  
Junbin Huang ◽  
Jinbo Peng ◽  
Fuhua Fei ◽  
...  
Keyword(s):  
Target Genes ◽  
Early Stage ◽  
Lamp Assay ◽  
Soft Rot ◽  
Isothermal Amplification ◽  
Comparative Genomic ◽  
Specific Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Accurate Detection ◽  
Specificity And Sensitivity

Bacterial soft rot caused by Pectobacterium species is a serious disease in konjac (Amorphophallus konjac), a healthy source of starch particularly in East Asia. An effective diagnostic method is crucial to control the disease and reduce losses in konjac production. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay with a specific primer set for the rapid and accurate detection of P. aroidearum. A comparative genomics approach was used to identify the specific genes suitable for the design of LAMP primers. The candidate target genes were determined through a first-round comparison with a 50-genome nucleotide database, and subjected to a second-round screening with the GenBank NR database. As a result, nine specific genes of P. aroidearum were selected for LAMP primer design. After screening of the primers, the primer set 1675-1 was chosen for LAMP detection owing to its high specificity and sensitivity. The LAMP assay could detect the presence of P. aroidearum genomic DNA at a concentration as low as 50 fg and 1.2 × 104 CFU/g artificially infected soil within 40 min at 65 °C. Subsequently, this primer set was successfully used to specifically detect P. aroidearum in naturally infected and non-symptomatic plant samples or soil samples from the field. This study indicates that a comparative genomic approach may facilitate the development of highly specific primers for LAMP assays, and a LAMP diagnostic assay with the specific primer set 1675-1 should contribute to the rapid and accurate detection of soft-rot disease in konjac at an early stage.

scholarly journals Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  
Keyword(s):  
Test Performance ◽  
Genomic Dna ◽  
Lamp Assay ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Genome Comparison ◽  
Performance Study ◽  
Loop Mediated Isothermal Amplification ◽  
Tilletia Controversa ◽  
Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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