scholarly journals Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 940
Author(s):  
Foteini Roumani ◽  
Sarah Azinheiro ◽  
Hugo Sousa ◽  
Ana Sousa ◽  
Mafalda Timóteo ◽  
...  
Keyword(s):  
Gold Standard ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Infected People ◽  
Loop Mediated Isothermal Amplification ◽  
Specialized Equipment ◽  
Good Concordance ◽  
One Year ◽  
Low Sensitivity

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  
Keyword(s):  
Real Time ◽  
Mycoplasma Pneumoniae ◽  
Rapid Detection ◽  
Clinical Laboratory ◽  
Direct Sequencing ◽  
Bacterial Species ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nasopharyngeal Swab ◽  
Loop Mediated Isothermal Amplification

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.

scholarly journals Loop-Mediated Isothermal Amplification Assay for Rapid and Reliable Detection of Mycobacterium tuberculosis in Sputum Samples

2018 ◽  
Vol 10 (2) ◽  
pp. 27-34
Author(s):  
B K Sharma ◽  
B D Pandey ◽  
K Sharma ◽  
B Sapkota ◽  
A Singh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Predictive Value ◽  
Gold Standard ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Positive Test ◽  
Fluorochrome Staining ◽  
Negative Test ◽  
Loop Mediated Isothermal Amplification ◽  
Thermal Cycler

Introduction: Tuberculosis (TB) remains a major global health problem. The most common method for diagnosing TB in developing countries is sputum smear microscopy; however, the sensitivity of this test is relatively lower. Detection of Mycobacterium tuberculosis using conventional culture and biochemical-based assays is time-consuming and laborious. Polymerase Chain Reaction (PCR) is also available for diagnosis of Mycobacterium tuberculosis. However, the PCR assay requires an expensive thermal cycler to amplify the DNA fragment in multiple temperature-dependent steps. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. The loop-mediated isothermal amplification (LAMP) assay is a diagnostic technique which can aid in the fight against TB in resource-poor countries. The LAMP assay can amplify a targeted sequence at a constant temperature. Therefore, a large and costly thermal cycler is not necessary for a LAMP assay.Objectives: The objective of this study was to identify Mycobacterium tuberculosis directly from sputum by LAMP and to compare its efficacy over routinely used methods.Methods: A total of 106 (53 fluorochrome staining positive and 53 fluorochrome staining negative) sputum samples were collected in this study. Mycobacterial DNA was extracted from concentrated sputum samples by freezing and boiling method. LAMP assay using a set of six specific primers targeting the M. tuberculosis 16S rRNA gene with high sensitivity was used to analyze sputum samples. The results were then compared with that of the culture method, which was considered as the gold standard method.Results: Among total of 106 samples studied by microscopy and culture, 53 were positive by both, whole four were positive by culture but negative by microscopy. With reference to culture, the microscopy had sensitivity 92.98%, specificity 100%, and predictive value of positive test 100%, predictive value of negative test 92.5%. Out of 106 samples subjected to culture and LAMP for the diagnosis of TB, 55 samples were positive by both tests and two were positive only in culture, while 48 were negative in both tests and one was negative only in culture. While comparing the LAMP with culture as a gold standard, the sensitivity of LAMP was 96.49%, specificity was 97.95%, predictive value of positive test was 98.21%, predictive value of negative test was 96%.Conclusions: Comparative experiments showed that the LAMP assay is a rapid, sensitive, and specific method to detect M. tuberculosis infection. Indeed, an inexpensive LAMP assay would be potential as a diagnostic test for tuberculosis, especially in resource-limited settings. J-GMC-N | Volume 11 | Issue 01 | January-June 2018, Page: 27-34

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 Development of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Phytopythium vexans

2021 ◽  
Vol 12 ◽  
Author(s):  
Tuhong Wang ◽  
Haojun Ji ◽  
Yongting Yu ◽  
Xiaojie Wang ◽  
Yi Cheng ◽  
...  
Keyword(s):  
Root Rot ◽  
Rapid Detection ◽  
Color Change ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Specialized Equipment ◽  
Root Tissues ◽  
And Control ◽  
Brown Root Rot

Brown root rot caused by Phytopythium vexans is a new destructive root disease on many plants such as Gingko, Citrus, kiwifruit, and ramie. The establishment of loop-mediated isothermal amplification (LAMP) technology for detecting P. vexans can help monitor and control brown root rot quickly, efficiently, and accurately. LAMP technology is known for its simplicity, sensitivity, and speed; and it does not require any specialized equipment – a water bath or a thermoblock is sufficient for isothermal amplifications. LAMP products can be visualized by using hydroxy naphthol blue (HNB) dye or agarose gel electrophoresis. In this study, by searching and comparing the internal transcribed spacer (ITS) sequences of P. vexans and the related species in oomycete genera Pythium, Phytopythium, and Phytophthora, we designed specific primers targeting the ITS gene region of P. vexans. Using HNB dye, we established a LAMP technique for rapid detection of P. vexans by visible color change. In addition, we optimized the protocol to enhance both sensitivity and specificity for P. vexans detection. Under the optimized condition, our protocol based on LAMP technology could detect as low as 24 copies of the P. vexans genomic DNA, which is ∼100 times more sensitive than conventional PCR. This method can successfully detect P. vexans using cell suspensions from P. vexans – infected ramie root tissues.

scholarly journals Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3

2022 ◽  
Vol 12 ◽  
Author(s):  
Jie Zhang ◽  
Miaomiao Li ◽  
Yunwen Ou ◽  
Danian Chen ◽  
Yaozhong Ding ◽  
...  
Keyword(s):  
Epidemiological Study ◽  
Mainland China ◽  
Porcine Circovirus Type ◽  
Isothermal Amplification ◽  
Porcine Circovirus ◽  
Lower Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
One Step ◽  
Type 3 ◽  
Taqman Qpcr

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017–2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.

scholarly journals LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) DALAM PENEGAKAN DIAGNOSIS INFEKSI PARASIT MALARIA BERBASIS MOLEKULER

2021 ◽  
Vol 9 (1) ◽  
pp. 120-129
Author(s):  
Jhons Fatriyadi Suwandi
Keyword(s):  
Nested Pcr ◽  
Gold Standard ◽  
Remote Area ◽  
Isothermal Amplification ◽  
Low Transmission ◽  
Loop Mediated Isothermal Amplification

Penegakan diagnosis malaria merupakan hal penting dalam program eleminasi malaria. Proses yang cepat, akurat dan murah merupakan harapan dalam diagnosis malaria, khususnya pada daerah remote area, pada kondisi gawat darurat atau pada saat ketidaktersediaan tenaga mikroskopis. Pemeriksaan RDT dan RDTs yang telah digunakan untuk menegakkan diagnosis malaria, terkadang memiliki akurasi yang kurang diharapkan. Pemeriksaan mikroskopis sebagai gold standard, sering terkendala saat tidak ada tenaga mikroskopis. Pengalaman tenaga mikroskopis juga akan mempengaruhi hasil pemeriksaan. Nested PCR sering digunakan untuk mengkonfirmasi hasil pemeriksaan mikroskopis, namun membutuhkan peralatan dan laboratorium khusus sehingga membutuhkan biaya yang mahal. Untuk menjawab kekurangan pada metode pemeriksaan sebelumnya, maka loop-mediated isothermal amplification (LAMP) dikembangkan sebagai metode diagnosis. LAMP sebagai metode pemeriksaan yang berbasis molekuler memiliki akurasi yang tinggi, dengan sensitifitas dan spesifisitas pada hampir semua penelitian sebelumnya lebih dari 90%. LAMP juga dapat mendeteksi spesies plasmodium dari pasien malaria pada daerah endemis atau non-endemis (kasus impor), pada kehamilan, pada kondisi densitas parasitemia yang rendah dan pada daerah dengan low transmission.

scholarly journals The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Mi-croorganisms: A Systematic Review and Meta-Analysis

2021 ◽  
Author(s):  
Yasaman Sadeghi ◽  
Pegah Kananizadeh ◽  
Solmaz Ohadian Moghadam ◽  
Ahad Alizadeh ◽  
Mohammad Reza Pourmand ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Likelihood Ratio ◽  
Gold Standard ◽  
Meta Analysis ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Chain Reaction ◽  
Cultivation Method ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Background: The loop-mediated isothermal amplification (LAMP) method is frequently used for identifying many microorganisms. The present review aimed to evaluate the sensitivity and specificity of LAMP method for detection of food-borne bacteria and to compare these features with those of polymerase chain reaction (PCR), as an alternative molecular diagnostic procedure, and with cultivation method, as the gold standard method. Methods: The literature was searched in electronic databases (PubMed, Scopus, Web of Science, and EMBASE) for recruiting publications within Jan 2000 to Jul 2021. We used the combinations of keywords including foodborne disease, LAMP, PCR, Loop-mediated isothermal amplification, and polymerase chain reaction. Meta-analysis was used to adjust the correlation and heterogeneity between the studies. The efficiency of the methods was presented by negative likelihood ratio, positive likelihood ratio, sensitivity, specificity, and odds ratio using forest plots. A P-value less than 0.05 was considered as statistical significance cut off. The confidence intervals were presented at the 95% interval. Results: Overall, 23 relevant studies were analyzed. The sensitivities of LAMP and PCR methods were estimated to be 96.6% (95% CI: 95.0-97.7) and 95.6% (95%CI: 91.5-97.8), respectively. The specificities of LAMP and PCR were also estimated to be 97.6% (95%CI: 92.6-99.3) and 98.7% (95%CI: 96.5-99.5), respectively. Conclusion: The specificities of LAMP and PCR assays were determined by comparing their results with cultivation method as the gold standard. Overall, the specificity of both PCR and LAMP methods was low for detection of fastidious bacteria. Nevertheless, LAMP and PCR methods have acceptable specificities and sensitivities, and their application in clinical practice necessitates more studies.

scholarly journals Dry loop mediated isothermal amplification assay for detection of SARS-CoV-2 from clinical specimens

2020 ◽  
Author(s):  
Yuki Higashimoto ◽  
Masaru Ihira ◽  
Yoshiki Kawamura ◽  
Masato Inaba ◽  
Kazuya Shirato ◽  
...  
Keyword(s):  
Developing Countries ◽  
Real Time ◽  
Predictive Value ◽  
Isothermal Amplification ◽  
Cold Chain ◽  
Pcr Analysis ◽  
Nasopharyngeal Swab ◽  
Lamp Method ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification

Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries.

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.

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