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.

Specific and sensitive detection of the guava fruit anthracnose pathogen (Colletotrichum gloeosporioides) by loop-mediated isothermal amplification (LAMP) assay

2020 ◽  
Vol 66 (1) ◽  
pp. 17-24
Author(s):  
Chengzhong Lan ◽  
Jinai Yao ◽  
Xiujuan Yang ◽  
Hongchun Ruan ◽  
Deyi Yu ◽  
...  
Keyword(s):  
Disease Management ◽  
Colletotrichum Gloeosporioides ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Guava Fruit ◽  
Disease Management Strategy ◽  
Highly Sensitive

Anthracnose of guava, caused by the fungus Colletotrichum gloeosporioides, is a major factor limiting worldwide guava production. Timely and accurate detection of the pathogen is important in developing a disease management strategy. Herein, a loop-mediated isothermal amplification (LAMP) assay for the specific and sensitive detection of C. gloeosporioides was developed using primers targeting the β-tubulin 2 (TUB2) gene. The optimal reaction conditions were 64 °C for 60 min. The specificity of the method was tested against C. gloeosporioides isolates, Colletotrichum spp. isolates, and isolates of other genera. Positive results were obtained only in the presence of C. gloeosporioides, whereas no cross-reaction was observed for other species. The detection limit of the LAMP assay was 10 fg of genomic DNA in a 25 μL reaction. The LAMP assay successfully detected C. gloeosporioides in guava fruit collected in the field. The results indicate that the developed LAMP assay is a simple, cost-effective, rapid, highly sensitive, and specific tool for the diagnosis of guava anthracnose caused by C. gloeosporioides and could be useful for disease management.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) diagnostic test for detection of whipworm, Trichuris trichiura, in faecal samples

2020 ◽  
Vol 94 ◽  
Author(s):  
M.G. Ngari ◽  
I.N. Mwangi ◽  
M.P. Njoroge ◽  
J. Kinyua ◽  
F.A. Osuna ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Infection Intensity ◽  
Lamp Assay ◽  
Trichuris Trichiura ◽  
Isothermal Amplification ◽  
Sub Saharan Africa ◽  
Health Concern ◽  
Alkaline Lysis ◽  
Loop Mediated Isothermal Amplification ◽  
Faecal Samples

Abstract Whipworm infection or trichuriasis caused by Trichuris trichiura is of major public health concern in sub-Saharan Africa, particularly among pre-school and school-going children. It is among the neglected tropical diseases targeted for elimination through mass drug administration (MDA). One of the outcomes of MDA is a rapid decline in levels of infection intensity, making it difficult to monitor effectiveness of control measures using the conventional Kato–Katz procedure, which relies on the microscopic detection of parasite ova in faecal samples. In the present study, a loop-mediated isothermal amplification (LAMP) test was developed for the detection of T. trichiura infection in faecal samples. LAMP technology offers greater sensitivity and specificity than the microscopy-based tests. A set of four specific primers targeting the internal transcribed spacer 2 region of the ribosomal DNA were designed using Primer Explorer software. DNA was extracted from faecal samples using the alkaline lysis method (HotSHOT) and the LAMP reaction performed at 63°C for 1 h. The amplicons were visualized by both gel electrophoresis and with the naked eye following staining with SYBR green dye. Sensitivity and specificity tests were determined using the standard Kato–Katz diagnostic procedure as a reference test. The developed LAMP assay reliably detected T. trichiura DNA in faecal samples, with a specificity and sensitivity of 88% and 77%, respectively. No cross-reactivity was observed with several common helminth parasites. The developed LAMP assay is an appropriate diagnostic method for the detection of T. trichiura DNA in human faecal samples due to its simplicity, low cost, high sensitivity and specificity.

scholarly journals Development of a reverse transcription loop mediated isothermal amplification assay for the detection of Mouse reovirus type 3 in laboratory mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taofeng Lu ◽  
Lingyun Tao ◽  
Haibo Yu ◽  
Hui Zhang ◽  
Yanjun Wu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Laboratory Mice ◽  
Blood Samples ◽  
S1 Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Reovirus Type ◽  
One Step ◽  
Type 3

AbstractMouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

scholarly journals A Novel Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification Detection Platform: Application to Diagnosis of COVID-19

2021 ◽  
Vol 9 ◽  
Author(s):  
Yi Wang ◽  
Xiaoxia Wang ◽  
Hai Chen ◽  
Limei Han ◽  
Licheng Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Virus Disease ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Health Concern ◽  
One Pot ◽  
Loop Mediated Isothermal Amplification

The ongoing Corona virus disease (COVID-19) outbreak has become a huge global health concern. Here, we reported a novel detection platform based on the loop-mediated isothermal amplification (LAMP), termed real-time reverse transcription LAMP (rRT-LAMP) and applied it for the diagnosis of COVID-19 (COVID-19 rRT-LAMP). rRT-LAMP integrates reverse transcription, LAMP amplification, restriction endonuclease cleavage and real-time fluorescence detection into one-pot reaction, and facilitates the diagnosis of COVID-19 at 64°C for only 35 min. The ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2 were detected for diagnosing COVID-19. The limit of detection (LoD) of COVID-19 rRT-LAMP assay was 14 copies (for each marker) per vessel, and no positive results were obtained from non-SARS-CoV-2 templates. To demonstrate its feasibility, a total of 33 oropharynx swab samples collected from COVID-19 patients also were diagnosed as SARS-CoV-2 infection using COVID-19 rRT-LAMP protocol. No cross-reactivity was yielded from 41 oropharynx swab samples collected from non-COVID-19 patients. These data suggesting that the COVID-19 rRT-LAMP assay is a potential detection tool for the diagnosis of SARS-CoV-2 infection in clinical, field and disease control laboratories, and will be valuable for controlling the COVID-19 epidemic.

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 Rapid diagnosis of Toxoplasma gondii using loop-mediated isothermal amplification assay in camels and small ruminants

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Gamil S. G. Zeedan ◽  
Abeer M. Abdalhamed ◽  
Raafat M. Shaapan ◽  
Amira H. El-Namaky
Keyword(s):  
Small Ruminants ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Blood Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Assay ◽  
Detection Capabilities

Abstract Background This study was conducted to detect the presence of T. gondii in milk and blood samples using three different assays: enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification assay (LAMP). Whole blood, serum, and milk samples were collected from goats (n = 156), sheep (n = 261), and camels (n = 108) in different governorates in Egypt from December 2019 to February 2021 and screened by ELISA for anti-Toxoplasma IgG antibodies before DNA extraction. The target T. gondii DNA gene was detected and evaluated using the LAMP assay compared to PCR. Results T. gondii antibodies were found in milk and serum samples at the rates of (29.26%) and (36.58%) in camels, (34.18%) and (35.89%) in sheep, and (33.7%) and (36.36%) in goats, respectively. Similar to PCR, the percentages of LAMP tests for the detection of the T. gondii DNA gene in milk and blood samples of camels, sheep, and goats were (4.8, 14.63), (6.83, 7.69), and (7.79, 9.09), respectively. LAMP's sensitivity for detecting T. gondii in milk and blood samples, which was identical to that of PCR, was 100%. Conclusions The findings clearly demonstrated that there were no variations in T. gondii detection capabilities in milk and blood samples from various animals using both PCR and LAMP tests. It provides a quick, precise, and sensitive method of detecting T. gondii in a variety of samples that may be used both in the field and in laboratory diagnosis.

scholarly journals Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9278 ◽  
Author(s):  
Yee Ling Lau ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
Meng Yee Lai ◽  
Tuan Suhaila Tuan Soh ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Resource Limited ◽  
Highly Sensitive ◽  
Comparable Performance

Background Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid. Methods A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection. Results This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

scholarly journals Loop-mediated isothermal amplification (LAMP) for rapid detection of L. monocytogenes in meat

10.5219/1165 ◽  
2019 ◽  
Vol 13 (1) ◽  
pp. 800-805
Author(s):  
Yuliya Yushina ◽  
Anzhelika Makhova ◽  
Elena Zayko ◽  
Dagmara Bataeva
Keyword(s):  
Foodborne Pathogens ◽  
Meat Products ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Meat Industry ◽  
Safety Testing ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Detection Assay ◽  
Highly Sensitive

There is a continued need to develop improved rapid methods for detection of foodborne pathogens. Rapid and sensitive methods for enumeration of Listeria monocytogenes are important for microbiological food safety testing purpose. The aim of this project was to evaluate a commercial loop-mediated isothermal amplification (LAMP) based system with bioluminescence, named as 3M™ Molecular Detection Assay (MDA), was validated for the detection of L. monocytogenes in food products with a standard GOST 32031-2012 method as reference. The results of this study revealed that a commercial LAMP-based method performed equally effective compared with method, showing from 94% to 100% specificity and sensitivity, respectively. The LAMP-based method was shown to be rapid and reliable detection technique for L. monocytogenes present at low numbers (10 CFU.g-1) on raw meat and meat products and can be applicable in meat industry. Thus, compared with the microbiological method based GOST 32031-2012, the LAMP assay is a relatively rapid and highly sensitive method for detecting L. monocytogenes and will facilitate the surveillance for contamination of L. monocytogenes in food. The 3M MDS result and culture-based detection (GOST 32031-2012) did not differ significantly (p >0.05) regarding the number of positive samples.

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