scholarly journals Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ryan Domingo ◽  
Cristian Perez ◽  
Diksha Klair ◽  
Huong Vu ◽  
Alika Candelario-Tochiki ◽  
...  
Keyword(s):  
Infected Plant ◽  
Field Tests ◽  
Lamp Assay ◽  
Soft Rot ◽  
Epidemiological Studies ◽  
Potato Industry ◽  
Isothermal Amplification ◽  
Specific Detection ◽  
Loop Mediated Isothermal Amplification

AbstractPectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

scholarly journals Detection and Control of Fusarium oxysporum from Soft Rot in Dendrobium officinale by Loop-Mediated Isothermal Amplification Assays

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1136
Author(s):  
Caiyun Xiao ◽  
Rongyu Li
Keyword(s):  
Fusarium Oxysporum ◽  
Integrated Management ◽  
Elongation Factor ◽  
Lamp Assay ◽  
Soft Rot ◽  
Isothermal Amplification ◽  
Dendrobium Officinale ◽  
Control Effect ◽  
Loop Mediated Isothermal Amplification ◽  
And Control

Soft rot causing Fusarium oxysporum is one of the most destructive diseases of Dendrobium officinale Kimura et Migo in China that reduces D. officinale yield and quality. A key challenge for an integrated management strategy for this disease is the rapid and accurate detection of F. oxysporum on D. officinale. Therefore, a new loop-mediated isothermal amplification (LAMP) assay was developed for this purpose. In this study, the primers were selected and designed using the translation elongation factor-1α (TEF-1α) gene region as the target DNA sequence in order to screen the best system of reaction of LAMP to detect F. oxysporum through optimizing different conditions of the LAMP reaction, including time, temperature, concentrations of MgSO4, and concentrations of inner and outer primers. The optimized system was able to efficiently amplify the target gene at 62 °C for 60 min with 1.2 μM internal primers, 0.4 μM external primers, 7 mM Mg2+, and 5 fg/µL minimum detection concentration of DNA for F. oxysporum. The amplified products could be detected with the naked eye after completion of the reaction with SYBR green I. We were better able to control the effect of soft rot in D. officinale using fungicides following a positive test result. Additionally, the control effect of synergism combinations against soft rot was higher than 75%. Thus, LAMP assays could detect F. oxysporum in infected tissues of D. officinale and soils in field, allowing for early diagnosis of the disease.

scholarly journals A Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Specific Detection of Airborne Inoculum of Uromyces betae (Sugar Beet Rust)

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 417-421 ◽  
Author(s):  
Agata M. Kaczmarek ◽  
Kevin M. King ◽  
Jonathan S. West ◽  
Mark Stevens ◽  
Debbie Sparkes ◽  
...  
Keyword(s):  
United Kingdom ◽  
Sugar Beet ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Specific Detection ◽  
Rust Disease ◽  
Loop Mediated Isothermal Amplification ◽  
The United Kingdom ◽  
Uromyces Betae ◽  
Forecasting System

Sugar beet rust disease (causal agent Uromyces betae) represents a serious threat to worldwide sugar beet (Beta vulgaris) crops, causing yield losses of up to 10% in the United Kingdom. Currently, the disease is managed mainly by application of fungicides after rust disease symptoms appear. Development of a future forecasting system, incorporating data on environmental factors and U. betae inoculum levels, would enable better disease control by more targeted application of fungicides. In this study, we developed a first molecular diagnostic, targeted to cytochrome b DNA sequences and based on loop-mediated isothermal amplification (LAMP) technology, for rapid (<30 min) and specific detection of U. betae. The new assay only detected U. betae strains (collected from across eastern England, the main sugar beet growing region in the United Kingdom) and Denmark; it did not detect other closely related pathogens (e.g., Puccinia sp., U. fabae) or others that are commonly found on sugar beet (Cercospora beticola, Erysiphe betae, Ramularia beticola). The assay could consistently detect down to small amounts of U. betae DNA (10 pg). Application of the new LAMP diagnostic to air spore tape samples collected between mid-June and mid-September from a single U.K. sugar beet field site revealed differences in temporal patterns of pathogen inoculum between the 2015 and 2016 seasons. The described LAMP assay could now be used as a component of a future automated inoculum-based forecasting system, enabling more targeted control of sugar beet rust disease.

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

2013 ◽  
Vol 142 (8) ◽  
pp. 1671-1677 ◽  
Author(s):  
M. KARANI ◽  
I. SOTIRIADOU ◽  
J. PLUTZER ◽  
P. KARANIS
Keyword(s):  
Lamp Assay ◽  
High Sensitivity ◽  
Leishmania Species ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Bench Scale ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range

SUMMARYWe developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

RAPID DIAGNOSIS OF BLUETONGUE VIRUS SEROTYPES 2 AND 12 INFECTION BY REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

2015 ◽  
Vol 41 (03) ◽  
pp. 187-196
Author(s):  
Jia-Ling Yang ◽  
Yi-Chia Li ◽  
Shu-Chia Hu ◽  
Fan Lee ◽  
Fun-In Wang
Keyword(s):  
Reverse Transcription ◽  
Bluetongue Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Universal Primers ◽  
Subclinical Infection ◽  
Loop Mediated Isothermal Amplification ◽  
Mouth Disease Virus ◽  
Field Samples

Bluetongue (BT), an arthropod-borne viral disease, is caused by the bluetongue virus (BTV), belonging to the genus Orbivirus of the family Reoviridae. Most species of ruminants are susceptible to BTV, but most infections go subclinical. These 'reservoir hosts' may potentially further increase the viral transmission and expansion of the disease; thus, detection of subclinical infection is important. To detect the BTV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed using primers targeted to six regions of the segment 5 (NS1) gene of the BTV2/KM/2003. The assay was completed in 1 h at a temperature 65°C, and the products were specifically digested with MboII enzyme presented in the target region. The in vitro sensitivity of the RT-LAMP was 100 copies, characterized by a qRT-PCR. The RT-LAMP did not cross-react with four tested common ruminant infectious agents, namely foot and mouth disease virus, goat pox virus, bovine herpesvirus 1, and Clostridium perfrigens. The RT-LAMP was applied to whole blood samples from 15 clinically healthy dairy cattle, and was able to detect BTV from 3/15 animals, and in particular 1 of the 3 animals was seronegative by cELISA. Positive RT-LAMP samples were reproducible. This RT-LAMP provides a simple, efficient, and sensitive method to specifically detect BTV and is suitable for the screening of field samples with a potential to pick up subclinical infection. The alignments of the outer primer region indicated matches of > 85% with 18 out of 26 BTV serotypes, implying its potential as universal primers.

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.

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