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.

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 Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

2020 ◽  
Vol 21 (21) ◽  
pp. 7981
Author(s):  
Catalina Avendaño ◽  
Manuel Alfonso Patarroyo
Keyword(s):  
Neglected Tropical Diseases ◽  
Point Of Care ◽  
Low Cost ◽  
High Specificity ◽  
World Health Organisation ◽  
Isothermal Amplification ◽  
World Health ◽  
Parasitic Infections ◽  
River Blindness ◽  
Loop Mediated Isothermal Amplification

The World Health Organisation (WHO) has placed twenty diseases into a group known as neglected tropical diseases (NTDs), twelve of them being parasitic diseases: Chagas’ disease, cysticercosis/taeniasis, echinococcosis, food-borne trematodiasis, human African trypanosomiasis (sleeping sickness), leishmaniasis, lymphatic filariasis, onchocerciasis (river blindness), schistosomiasis, soil-transmitted helminthiasis (ascariasis, hookworm, trichuriasis), guinea-worm and scabies. Such diseases affect millions of people in developing countries where one of the main problems concerning the control of these diseases is diagnosis-based due to the most affected areas usually being far from laboratories having suitable infrastructure and/or being equipped with sophisticated equipment. Advances have been made during the last two decades regarding standardising and introducing techniques enabling diagnoses to be made in remote places, i.e., the loop-mediated isothermal amplification (LAMP) technique. This technique’s advantages include being able to perform it using simple equipment, diagnosis made directly in the field, low cost of each test and the technique’s high specificity. Using this technique could thus contribute toward neglected parasite infection (NPI) control and eradication programmes. This review describes the advances made to date regarding LAMP tests, as it has been found that even though several studies have been conducted concerning most NPI, information is scarce for others.

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.

scholarly journals Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study

2020 ◽  
Author(s):  
Azeem Mehmood Butt ◽  
Shafiqa Siddique ◽  
Xiaoping An ◽  
Yigang Tong
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification ◽  
Qualitative Detection ◽  
Detection Assay ◽  
Testing Protocols ◽  
Positive Results

AbstractSevere acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as a rapidly spreading global pathogen stressing the need for development of rapid testing protocols ever than before. The aim of present study was to develop a SARS-CoV-2 detection protocol which can be performed within minimal resources and timeframe. For this purpose, we implemented the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology for the qualitative detection of SARS-CoV-2 RNA. In order to improve the detection capability, the RT-LAMP assay was developed to simultaneously amplify two viral genes: ORF1a and N. A total of 45 SARS-CoV-2 associated coronavirus disease 2019 (COVID-19) and 25 non-COVID-19 cases were enrolled. Viral RNA was extracted from the nasopharyngeal swab samples and analyzed simultaneously using PCR and RT-LAMP protocols. Overall, our SARS-CoV-2 dual gene RT-LAMP assay was found to be 95% accurate in detecting positive cases and showed no cross-reactivity or false-positive results in non-COVID-19 samples. Further evaluation on larger and multi-centric cohorts is currently underway to establish the diagnostic accuracy and subsequent implementation into clinical practice and at point-of-care settings.

scholarly journals Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1629
Author(s):  
Alexander Domnich ◽  
Andrea Orsi ◽  
Donatella Panatto ◽  
Vanessa De Pace ◽  
Valentina Ricucci ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Point Of Care ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Loop Mediated Isothermal Amplification

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

scholarly journals Rapid And Sensitive Detection Of Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification

2015 ◽  
Vol 65 (1) ◽  
pp. 20-29 ◽  
Author(s):  
PARK Byung-Yong ◽  
SHIM Kwan-Seob ◽  
KIM Won-Il ◽  
HOSSAIN Md Mukter ◽  
KIM Bumseok ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Sensitive Detection ◽  
Detection Methods ◽  
Conventional Pcr ◽  
Fecal Samples ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop

Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

scholarly journals Field Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Platform for the Detection of Schistosoma japonicum Infection in Oncomelania hupensis Snails

2018 ◽  
Vol 3 (4) ◽  
pp. 124 ◽  
Author(s):  
Zhi-Qiang Qin ◽  
Jing Xu ◽  
Ting Feng ◽  
Shan Lv ◽  
Ying-Jun Qian ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Schistosoma Japonicum ◽  
Point Of Care ◽  
Infected Snail ◽  
Lamp Assay ◽  
Field Evaluation ◽  
Isothermal Amplification ◽  
Pcr Analysis ◽  
Schistosomiasis Control ◽  
Loop Mediated Isothermal Amplification

Schistosoma infection in snails can be monitored by microscopy or indirectly by sentinel mice. As both these approaches can miss infections, more sensitive tests are needed, particularly in low-level transmission settings. In this study, loop-mediated isothermal amplification (LAMP) technique, designed to detect a specific 28S ribosomal Schistosoma japonicum (Sj28S) gene with high sensitivity, was compared to microscopy using snail samples from 51 areas endemic for schistosomiasis in five Chinese provinces. In addition, the results were compared with those from polymerase chain reaction (PCR) by adding DNA sequencing as a reference. The testing of pooled snail samples with the LAMP assay showed that a dilution factor of 1/50, i.e., one infected snail plus 49 non-infected ones, would still result in a positive reaction after the recommended number of amplification cycles. Testing a total of 232 pooled samples, emanating from 4006 snail specimens, showed a rate of infection of 6.5%, while traditional microscopy found only 0.4% positive samples in the same materials. Parallel PCR analysis confirmed the diagnostic accuracy of the LAMP assay, with DNA sequencing even giving LAMP a slight lead. Microscopy and the LAMP test were carried out at local schistosomiasis-control stations, demonstrating that the potential of the latter assay to serve as a point-of-care (POC) test with results available within 60–90 min, while the more complicated PCR test had to be carried out at the National Institute of Parasitic Diseases (NIPD) in Shanghai, China. In conclusion, LAMP was found to be clearly superior to microscopy and as good as, or better than, PCR. As it can be used under field conditions and requires less time than other techniques, LAMP testing would improve and accelerate schistosomiasis control.

scholarly journals ASarcoptes scabieispecific isothermal amplification assay for detection of this important ectoparasite of wombats and other animals

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5291 ◽  
Author(s):  
Tamieka A. Fraser ◽  
Scott Carver ◽  
Alynn M. Martin ◽  
Kate Mounsey ◽  
Adam Polkinghorne ◽  
...  
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Sarcoptes Scabiei ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Specific Pcr ◽  
Animal Populations ◽  
Thermal Cycler ◽  
Low Sensitivity ◽  
Species Specific

BackgroundThe globally distributed epidermal ectoparasite,Sarcoptes scabiei,is a serious health and welfare burden to at-risk human and animal populations. Rapid and sensitive detection ofS. scabieiinfestation is critical for intervention strategies. While direct microscopy of skin scrapings is a widely utilised diagnostic method, it has low sensitivity. PCR, alternatively, has been shown to readily detect mite DNA even in microscopy-negative skin scrapings. However, a limitation to the latter method is the requirements for specialised equipment and reagents. Such resources may not be readily available in regional or remote clinical settings and are an important consideration in diagnosis of this parasitic disease.MethodologyA Loop Mediated Isothermal Amplification (LAMP) assay targeting the ITS-2 gene forS. scabieiwas developed and evaluated on clinical samples from various hosts, previously screened with conventionalS. scabies-specific PCR. Species specificity of the newly developed LAMP assay was tested against a range of DNA samples from other arthropods. The LAMP assays were performed on a real-time fluorometer as well as thermal cycler to evaluate an end-point of detection. Using skin scrapings, a rapid sample processing method was assessed to eliminate extensive processing times involved with DNA extractions prior to diagnostic assays, including LAMP.ResultsTheS. scabieiLAMP assay was demonstrated to be species-specific and able to detect DNA extracted from a single mite within a skin scraping in under 30 minutes. Application of this assay to DNA extracts from skin scrapings taken from a range of hosts revealed 92.3% congruence (with 92.50% specificity and 100% sensitivity) to the conventional PCR detection ofS. scabiei. Preliminary results have indicated that diagnostic outcome from rapidly processed dry skin scrapings using our newly developed LAMP is possible in approximately 40 minutes.DiscussionWe have developed a novel, rapid and robust molecular assay for detectingS. scabieiinfesting humans and animals. Based on these findings, we anticipate that this assay will serve an important role as an ancillary diagnostic tool at the point-of-care, complementing existing diagnostic protocols forS. scabiei.

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