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 an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

2008 ◽  
Vol 57 (4) ◽  
pp. 439-443 ◽  
Author(s):  
Basu Dev Pandey ◽  
Ajay Poudel ◽  
Tomoko Yoda ◽  
Aki Tamaru ◽  
Naozumi Oda ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Rrna Gene ◽  
Predictive Values ◽  
Loop Mediated Isothermal Amplification ◽  
System A

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5–97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

scholarly journals Loop-Mediated Isothermal Amplification Assay for the Rapid Detection ofStaphylococcus aureus

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
King Ting Lim ◽  
Cindy Shuan Ju Teh ◽  
Kwai Lin Thong
Keyword(s):  
Predictive Value ◽  
Food Poisoning ◽  
Lamp Assay ◽  
Rapid Identification ◽  
Negative Control ◽  
Isothermal Amplification ◽  
Promising Alternative ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Wide Range

Staphylococcus aureus, including methicillin-resistantS. aureus(MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting thearcCgene ofS. aureuswas developed and evaluated with 119S. aureusand 25 non-S. aureusstrains. The usefulness of the assay was compared with the PCR method that targetsspaandarcCgenes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spaandarcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureusand one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification ofS. aureus. The LAMP assay is a promising alternative method for the rapid identification ofS. aureusand could be used in resource-limited laboratories and fields.

scholarly journals Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum

1970 ◽  
Vol 7 (2) ◽  
pp. 109-114 ◽  
Author(s):  
A Poudel ◽  
BD Pandey ◽  
B Lekhak ◽  
B Rijal ◽  
BR Sapkota ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Common Symptom ◽  
Bcg Vaccination ◽  
Loop Mediated Isothermal Amplification ◽  
16S Rna ◽  
Clinical Profiling

Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. Objectives: The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Materials and methods: Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Result: Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (Χ2=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. Conclusion: As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal. Key words: clinical profiling; Sputum; DNA; LAMP; M. tuberculosis; Nepal DOI: 10.3126/kumj.v7i2.2701 Kathmandu University Medical Journal (2009) Vol.7, No.2 Issue 26, 109-114

scholarly journals Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244753
Author(s):  
Jeeyong Kim ◽  
Borae G. Park ◽  
Da Hye Lim ◽  
Woong Sik Jang ◽  
Jeonghun Nam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Positive Reaction ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Published Data ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

scholarly journals Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinggui Yang ◽  
Junfei Huang ◽  
Xu Chen ◽  
Ziyu Xiao ◽  
Xiaojuan Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Clinical Specimen ◽  
Lamp Assay ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Whole Process ◽  
Specimen Processing ◽  
Mycobacterium Africanum

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66°C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 × 103 CFU/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1–2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.

scholarly journals Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia

2020 ◽  
Vol 8 (1) ◽  
pp. 103 ◽  
Author(s):  
Andrea Vergara ◽  
Hervé Boutal ◽  
Adrián Ceccato ◽  
Míriam López ◽  
Adrià Cruells ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Hospital Acquired Pneumonia ◽  
Hospital Acquired

Rapid identification of the causative agent of hospital-acquired pneumonia (HAP) will allow an earlier administration of a more appropriate antibiotic and could improve the outcome of these patients. The aim of this study was to develop a rapid protocol to identify the main microorganisms involved in HAP by loop-mediated isothermal amplification (LAMP) directly from respiratory samples. First of all, a rapid procedure (<30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A specific LAMP for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was performed with the extracted solution at 65 °C for 30–40 min. Overall, 58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to the microorganism detected. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%) compared with culture. Meanwhile for BAS/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. The turnaround time including sample preparation and LAMP was circa 1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.

scholarly journals Characterization of the tufB-secE-nusG-rplKAJL-rpoB Gene Cluster of the Citrus Greening Organism and Detection by Loop-Mediated Isothermal Amplification

Plant Disease ◽  
2005 ◽  
Vol 89 (7) ◽  
pp. 705-711 ◽  
Author(s):  
Mitsuru Okuda ◽  
Mitsuhito Matsumoto ◽  
Yuko Tanaka ◽  
Siti Subandiyah ◽  
Toru Iwanami
Keyword(s):  
Gene Cluster ◽  
Lamp Assay ◽  
Rpob Gene ◽  
Complete Sequence ◽  
Isothermal Amplification ◽  
Citrus Greening ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Conserved Sequence ◽  
Thermal Cycler

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to amplify the uncharacterized regions adjacent to the nusG-rplKAJL-rpoB gene cluster of citrus greening organism (GO) isolates from different locations in Japan and Indonesia. Conventional PCR was used to amplify the internal nusG-rplKAJL-rpoB gene cluster of these isolates, and the complete sequence of this 6.1-kb fragment was determined. Comparisons with other bacterial sequences showed that the fragment is the tufB-secE-nusG-rplKAJL-rpoB gene cluster. The organization of this gene cluster is similar to that of the homologous cluster found in Escherichia coli. Except for three nucleotide changes, the sequence was identical among Japanese and Indonesian isolates. A loop-mediated isothermal amplification (LAMP) assay based on the conserved sequence of the nusG-rplKAJL-rpoB gene cluster was developed for the detection of the GO. The LAMP product was rapidly detected on nylon membranes by staining with AzurB. LAMP could detect as low as about 300 copies of the nusG-rplKAJL-rpoB fragment of the Japanese and Indonesian isolates of GO. The LAMP-based detection method, which does not depend upon a thermal cycler and electrophoresis apparatus, will be useful for under-equipped laboratories, including those found in extension centers and quarantine offices.

scholarly journals Assessment of a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic bacteria from respiratory samples in patients with hospital-acquired pneumonia

2019 ◽  
Author(s):  
Andrea Vergara ◽  
Hervé Boutal ◽  
Adrián Ceccato ◽  
Míriam López ◽  
Adrià Cruells ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Hospital Acquired Pneumonia ◽  
Hospital Acquired

AbstractIntroductionHospital-acquired pneumonia (HAP) is the one that presents clinically two or more days after admission into the hospital. Rapid identification of the causative agent of HAP will allow an earlier administration of a more appropriate antibiotic therapy and could lead to an improved outcome of patients with HAP.MethodsFirst of all, a rapid procedure (< 30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A loop-mediated isothermal amplification reaction (LAMP) specific for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was carried out with the extracted solution. The reaction was performed at 65ºC for 30-40 min. LAMP was compared with bacterial culture method.ResultsOverall, 58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to the microorganism detected and to the respiratory sample analyzed. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%). Meanwhile for BAS/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. These scores were obtained including minor errors as correct. The turnaround time including preparation of the sample and LAMP was circa 1 hour.ConclusionsLAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.

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