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 Loop Mediated Isothermal Amplification for Diagnosis ofAscaris lumbricoidesin Fecal Samples

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Esther A. Shiraho ◽  
Agola L. Eric ◽  
Ibrahim N. Mwangi ◽  
Geoffrey M. Maina ◽  
Joseph M. Kinuthia ◽  
...  
Keyword(s):  
Neglected Tropical Diseases ◽  
Point Of Care ◽  
Infection Intensity ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Alkaline Lysis ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
The Common ◽  
Low Sensitivity

Ascaris lumbricoidesis a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers.Ascarisadult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detectedAscarisDNA from a single egg and in fecal samples. The assay specifically detectedAscarisDNA without amplifying DNA from ova of other parasites which commonly coexist withA. lumbricoidesin feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.

scholarly journals Colorimetric Reverse Transcription–Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2

2021 ◽  
Author(s):  
Meng Yee Lai ◽  
Soo Nee Tang ◽  
Yee Ling Lau
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Virus Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Detection Technology ◽  
Simple Detection

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.

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.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for diphtheria

2019 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

2020 ◽  
Author(s):  
Manju Singh ◽  
Saurabh Gupta ◽  
Shoor Vir Singh ◽  
Gururaj Kumaresan ◽  
Deepansh Sharma ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Lamp Assay ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Isothermal Amplification ◽  
Assay Method ◽  
Loop Mediated Isothermal Amplification ◽  
Is900 Pcr

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

scholarly journals Validation of the loop-mediated isothermal amplification method for rapid and sensitive detection of Ureaplasma species in respiratory tracts of preterm infants

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247618
Author(s):  
Yuta Mikami ◽  
Kazumasa Fuwa ◽  
Eriko Arima ◽  
Yasuo Suda ◽  
Itaru Yanagihara ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Preterm Infants ◽  
Sensitivity And Specificity ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Tracheal Aspirates

Introduction A simple and rapid diagnosis of Ureaplasma spp. is required for the choice of the appropriate antibiotic. However, an ideal detection method has not been available. This study examines the efficacy of the loop-mediated isothermal amplification (LAMP) assay, which provides rapid and sensitive results, to detect Ureaplasma spp. in respiratory tract samples of preterm infants. Methods The study included preterm infants born before 32 weeks of gestation admitted Kagoshima City Hospital from June 2018 to March 2020. Nasopharyngeal swabs and/or tracheal aspirates were obtained in the first seven postnatal days. One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were analyzed by LAMP, culture, and quantitative real-time polymerase chain reaction. Results All 167 infants had a median (range) gestational age of 28.7 weeks (22.3–30.9) and birthweight 1030g (322–1828). One hundred sixty-seven nasopharyngeal swabs and 101 tracheal aspirates were obtained. In the results of nasopharyngeal swabs, the sensitivity and specificity of LAMP were 73.9% (17/23) and 97.2% (140/144), whereas those of quantitative real-time polymerase chain reaction were 73.9% (17/23) and 95.8% (138/144), compared to culture. In the results of tracheal aspirates, the sensitivity and specificity of LAMP were 89.5% (17/19) and 92.7% (76/82), whereas those of quantitative real-time polymerase chain reaction were 89.5% (17/19) and 93.9% (77/82), compared to culture. Conclusions The LAMP assay showed similar sensitivity and specificity with quantitative real-time polymerase chain reaction in the respiratory tracts of preterm infants including extremely preterm infants during the immediate postnatal period. Therefore, the LAMP is a practical alternative for the early detection so that appropriate antibiotics can be administered for preventing BPD.

scholarly journals Loop-Mediated Isothermal Amplification Assay for Detecting Tumor Markers and Human Papillomavirus: Accuracy and Supplemental Diagnostic Value to Endovaginal MRI in Cervical Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Benjamin Wormald ◽  
Jesus Rodriguez-Manzano ◽  
Nicolas Moser ◽  
Ivana Pennisi ◽  
Thomas E. J. Ind ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Tumor Marker ◽  
Tumor Markers ◽  
Sensitivity And Specificity ◽  
Cancer Detection ◽  
Early Stage ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

ObjectiveTo establish the sensitivity and specificity of a human papillomavirus (HPV) and tumor marker DNA/mRNA assay for detecting cervical cancer that is transferrable to a Lab-on-a-chip platform and determine its diagnostic benefit in early stage disease when used in conjunction with high-resolution endovaginal magnetic resonance imaging (MRI).MethodsForty-one patients (27 with Stage1 cervical cancer [Group1] and 14 non-cancer HPV negative controls [Group2]) had DNA and RNA extracted from cervical cytology swab samples. HPV16, HPV18, hTERT, TERC/GAPDH and MYC/GAPDH concentration was established using a loop mediated isothermal amplification (LAMP) assay. Thresholds for tumor marker detection for Group1 were set from Group2 analysis (any hTERT, TERC/GAPDH 3.12, MYC/GAPDH 0.155). Group 1 participants underwent endovaginal MRI. Sensitivity and specificity for cancer detection by LAMP and MRI individually and combined was documented by comparison to pathology.ResultsSensitivity and specificity for cancer detection was 68.8% and 77.8% if any tumor marker was positive regardless of HPV status (scenario1), and 93.8% and 55.8% if tumor marker or HPV were positive (scenario 2). Adding endovaginal MRI improved specificity to 88.9% in scenario 1 (sensitivity 68.8%) and to 77.8%% in scenario2 (sensitivity 93.8%).ConclusionSpecificity for cervical cancer detection using a LAMP assay is superior with tumor markers; low sensitivity is improved by HPV detection. Accuracy for early stage cervical cancer detection is optimal using a spatially multiplexed tumor marker/HPV LAMP assay together with endovaginal MRI.

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