scholarly journals Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)

2018 ◽  
Vol 12 (11) ◽  
pp. e0006922 ◽  
Author(s):  
Keerti Kaumudee Dixit ◽  
Sandeep Verma ◽  
Om Prakash Singh ◽  
Dharmendra Singh ◽  
Akhil Pratap Singh ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Lamp Assay ◽  
Sybr Green ◽  
Isothermal Amplification ◽  
Sybr Green I ◽  
Loop Mediated Isothermal Amplification ◽  
Closed Tube ◽  
Blood Lysis

scholarly journals Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Daniel Moreira de Avelar ◽  
Débora Moreira Carvalho ◽  
Ana Rabello
Keyword(s):  
Visceral Leishmaniasis ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Health Concern ◽  
Serological Tests ◽  
Blood Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Human Visceral Leishmaniasis ◽  
Highly Sensitive ◽  
Sensitivity Specificity

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.

Simplified closed tube loop mediated isothermal amplification (LAMP) assay for visual diagnosis of Leishmania infection

Acta Tropica ◽  
2020 ◽  
Vol 212 ◽  
pp. 105651 ◽  
Author(s):  
Charanyarut Sukphattanaudomchoke ◽  
Suradej Siripattanapipong ◽  
Thanyapit Thita ◽  
Saovanee Leelayoova ◽  
Phunlerd Piyaraj ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Leishmania Infection ◽  
Loop Mediated Isothermal Amplification ◽  
Closed Tube ◽  
Visual Diagnosis

scholarly journals Validation of SYBR green I based closed‐tube loop‐mediated isothermal amplification (LAMP) assay for diagnosis of knowlesi malaria

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Meng Yee Lai ◽  
Choo Huck Ooi ◽  
Yee Ling Lau
Keyword(s):  
Nested Pcr ◽  
Plasmodium Knowlesi ◽  
Lamp Assay ◽  
Health Department ◽  
Sybr Green ◽  
Sybr Green I ◽  
Lamp Method ◽  
Blood Samples ◽  
Closed Tube ◽  
Colour Changes

Abstract Background As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi. Methods A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. Results LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. Conclusions These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

scholarly journals Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria

Diagnostics ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1689
Author(s):  
Mudsser Azam ◽  
Kirti Upmanyu ◽  
Ratan Gupta ◽  
Karugatharayil Sasi Sruthy ◽  
Monika Matlani ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Plasmodium Vivax ◽  
Sensitivity And Specificity ◽  
Limit Of Detection ◽  
Lamp Assay ◽  
Repeat Sequence ◽  
Isothermal Amplification ◽  
Malaria Surveillance ◽  
Loop Mediated Isothermal Amplification ◽  
Closed Tube

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

scholarly journals Utility of the Loop-Mediated Isothermal Amplification Assay for the Diagnosis of Visceral Leishmaniasis from Blood Samples in Ethiopia

2021 ◽  
Author(s):  
Dawit Gebreegzabher Hagos ◽  
Yazezew Kebede Kros ◽  
Mahmud Abdulkader ◽  
Zekarias Gessessew Arefaine ◽  
Etsay Nigus ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Lamp Assay ◽  
Accurate Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Assay ◽  
Loop Mediated Isothermal Amplification ◽  
Endemic Setting ◽  
Prompt Treatment ◽  
Amplification Assay ◽  
Hiv 1

Rapid and accurate diagnosis of visceral leishmaniasis (VL) is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69–98.66) and a specificity of 98.0% (95% CI: 94.34–99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61–98.34) in HIV-negatives and 100% (95% CI: 85.18–100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10–98.11) of the microscopy+ cases and was negative in 11/27 (40.7%, 95% CI: 22.39–61.20) of the microscopy− cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35–97.62) of the rK39+ cases and was negative in 149/154 (96.8%, 95% CI: 92.59–98.94) of the rK39− cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64–98.73) of the microscopy+ cases and negative in only 12/27 (44.4%, 95% CI: 25.48–64.67) of the microscopy– cases. There was an excellent agreement between rK39 and LAMP (Kappa = 0.91, 95% CI: 0.86–0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52–99.89) and a specificity of 98.0% (95% CI: 94.34–99.59). The findings demonstrate that LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 coinfected patients, in an endemic setting.

scholarly journals Detection of Phytophthora infestans by Loop-Mediated Isothermal Amplification, Real-Time LAMP, and Droplet Digital PCR

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 708-716 ◽  
Author(s):  
Jean B. Ristaino ◽  
Amanda C. Saville ◽  
Rajesh Paul ◽  
Donald C. Cooper ◽  
Qingshan Wei
Keyword(s):  
Phytophthora Infestans ◽  
Real Time ◽  
Detection Threshold ◽  
Lamp Assay ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Sybr Green ◽  
Isothermal Amplification ◽  
Conventional Pcr ◽  
Loop Mediated Isothermal Amplification

Phytophthora infestans is the causal agent of potato late blight, a devastating disease of tomato and potato and a threat to global food security. Early detection and intervention is essential for effective management of the pathogen. We developed a loop-mediated isothermal amplification (LAMP) assay for P. infestans and compared this assay to conventional PCR, real-time LAMP, and droplet digital PCR for detection of P. infestans. The LAMP assay was specific for P. infestans on potato and tomato and did not amplify other potato- or tomato-infecting Phytophthora species or other fungal and bacterial pathogens that infect potato and tomato. The detection threshold for SYBR Green LAMP and real-time LAMP read with hydroxynaphthol blue and EvaGreen was 1 pg/µl. In contrast, detection by conventional PCR was 10 pg/µl. Droplet digital PCR had the lowest detection threshold (100 fg/µl). We adapted the LAMP assay using SYBR Green and a mobile reader (mReader) for use in the field. Detection limits were 584 fg/µl for SYBR Green LAMP read on the mReader, which was more sensitive than visualization with the human eye. The mobile platform records geospatial coordinates and data from positive pathogen detections can be directly uploaded to a cloud database. Data can then be integrated into disease surveillance networks. This system will be useful for real-time detection of P. infestans and will improve the timeliness of reports into surveillance systems such as USABlight or EuroBlight.

scholarly journals Выявление мутации Ser450Leu в гене rpoB Mycobacterium tuberculosis методом аллель-специфичной изотермической петлевой амплификации ДНК

2019 ◽  
pp. 36-43
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sybr Green ◽  
Isothermal Amplification ◽  
Sybr Green I ◽  
Loop Mediated Isothermal Amplification

Для выявления генетических мутаций широко используют достаточно трудоемкий и дорогостоящий метод полимеразной цепной реакции (ПЦР). Целью работы было оценить возможность применения двух схем метода аллель-специфичной изотермической петлевой амплификации (loop-mediated isothermal amplification, LAMP) для выявления мутации TCG/TTG (S450L) в гене rpoB Mycobacterium tuberculosis. Использовали 48 клинических изолятов M. tuberculosis и 11 образцов мокроты, выбранных случайным образом и полученных в микробиологической лаборатории г. Новосибирска от пациентов с впервые выявленным заболеванием. Показано, что применение схемы анализа с использованием аллель-специфичного праймера FIP по сравнению с F3 имеет лучшую разрешающую способность: разница между временем амплификации мутации и аллеля дикого типа составила 22 ± 2,4 против 13 ± 4,1 мин (p = 0,0011). При использовании 100 геном-эквивалентов ДНК истинно положительный сигнал (амплификация гена rpoB с мутацией при использовании соответствующего аллель-специфического праймера) детектировался после 29,4 ± 3,4 мин. Положительный сигнал визуализировался после добавления в реакцию SYBR Green I, как при освещении дневным светом, так и при использовании трансиллюминатора с УФ-излучением. С помощью разработанного нами метода была проанализирована выборка ДНК 20 RIFR изолятов M. tuberculosis, несущих мутацию Ser450Leu в гене rpoB, 10 RIFR изолятов, несущих другие мутации в гене rpoB, а также 18 RIFs изолятов без мутаций; наличие мутаций в образцах было определено с помощью классического секвенирования по Сенгеру. Чувствительность и специфичность LAMP для выявления мутации Ser450Leu в гене rpoB составили 100%. Данный подход позволяет использовать в качестве ДНК грубые лизаты микобактерий, что сокращает тотальное время анализа до 1,5 ч.

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