scholarly journals Loop-Mediated Isothermal Amplification for the Rapid Detection of the F200Y Mutant Genotype of Carbendazim-Resistant Isolates of Sclerotinia sclerotiorum

Plant Disease ◽  
2016 ◽  
Vol 100 (5) ◽  
pp. 976-983 ◽  
Author(s):  
Yabing Duan ◽  
Ying Yang ◽  
Yong Wang ◽  
Xiayan Pan ◽  
Jian Wu ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Special Equipment ◽  
Mutant Genotype ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results ◽  
Resistant Mutants ◽  
Moderate Resistance

The point mutation at codon 200 (TTC→TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63°C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.

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.

Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens

2019 ◽  
Author(s):  
Maryam ARFAATABAR ◽  
Narjes NOORI GOODARZI ◽  
Davoud AFSHAR ◽  
Hamed MEMARIANI ◽  
Ghasem AZIMI ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Respiratory Specimens

  Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clini-cal specimens collected from patients with pneumonia. Methods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined. Results: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ~ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (κ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the con-gruence between LAMP assay and PCR assay was ‘almost perfect’ (κ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR. Conclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.

scholarly journals Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Erwinia amylovora on Pear and Apple Fruit Flowers

Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 423-430 ◽  
Author(s):  
Todd N. Temple ◽  
Kenneth B. Johnson
Keyword(s):  
Rapid Detection ◽  
Pathogen Detection ◽  
Fire Blight ◽  
Management Practices ◽  
Erwinia Amylovora ◽  
Lamp Assay ◽  
Apple Fruit ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Forecasting Models

Fire blight of pear and apple is frequently an inoculum-limited disease but weather-based forecasting models commonly assume that the pathogen is omnipresent. To improve fire blight risk assessment during flowering, we developed a rapid pathogen detection protocol that uses loop-mediated isothermal amplification (LAMP) to detect DNA of epiphytic Erwinia amylovora on samples of pear and apple flowers. LAMP detected a single flower colonized epiphytically by E. amylovora in a sample of 100 flower clusters (approximately 600 flowers). Samples of 100 flower clusters from orchards (approximately one sample per hectare) were washed and subjected to LAMP, which was completed in 2 h. In three experimental orchards inoculated with E. amylovora, positive LAMP reactions were attained from nine of nine 100-flower cluster samples; pathogen populations in the floral washes averaged 5.2 × 103 CFU per flower as determined by dilution plating. Samples of pear and apple flowers obtained from 60 commercial orchards located in Oregon, Washington, California, and Utah resulted in detection of E. amylovora by LAMP assay from 34 sites, 20 of which developed fire blight. Of samples at early bloom, 10% were positive for epiphytic E. amylovora compared with 28% at petal fall; pathogen density in washes of positive samples averaged 3.2 × 102 CFU per flower. In another 26 orchards, all floral washes were negative for E. amylovora by LAMP and by dilution plating; a light severity of fire blight was observed in 8 of these orchards. Overall, positive detection of epiphytic E. amylovora in commercial orchards by LAMP-based scouting generally occurred at later stages of bloom after heat (risk) units had begun to accumulate, an indication that weather-based forecasting models may be an adequate measure of fire blight risk for many orchardists. Nonetheless, several orchardists communicated that information from the LAMP-based rapid detection protocol resulted in modification of their fire blight management practices.

Sign in / Sign up

Export Citation Format

Share Document