Closed-tube Loop-mediated Isothermal Amplification Research Articles

Rapid Visual Detection of Methicillin-Resistant Staphylococcus aureus in Human Clinical Samples via Closed LAMP Assay Targeting mecA and spa Genes.

The emergence of antimicrobial resistance (AMR), particularly methicillin-resistant Staphylococcus aureus (MRSA), poses a significant global health threat as these bacteria increasingly become resistant to the most available therapeutic options. Thus, developing an efficient approach to rapidly screen MRSA directly from clinical specimens has become vital. In this study, we establish a closed-tube loop-mediated isothermal amplification (LAMP) method incorporating hydroxy-naphthol blue (HNB) colorimetric dye assay to directly detect MRSA from clinical samples based on the presence of mecA and spa genes. In total, 125 preidentified S. aureus isolates and 93 clinical samples containing S. aureus were sourced from the microbiology laboratory at Hamad General Hospital (HGH). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were computed based on conventional PCR. The assay demonstrated 100% specificity, 91.23% sensitivity, 0.90 Cohen Kappa (CK), 100% PPV, and 87.8% NPV for the clinical samples, while clinical isolates exhibited 100% specificity, 97% sensitivity, 0.926 CK, 100% PPV, and 88.89% NPV. Compared to cefoxitin disk diffusion, LAMP provided 100% specificity and sensitivity, 1.00 CK, and 100% for PPV and NPV. The study revealed that the closed-tube LAMP incorporating (HNB) dye is a rapid technique with a turnaround time of less than 1 h and high specificity and sensitivity.

A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T.

Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Rapid visualization and detection of Staphylococcus aureus based on loop-mediated isothermal amplification.

Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 14 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by tenfold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP (test accuracy, 99.46%). The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

A high sensitivity method of closed-tube loop-mediated isothermal amplification developed for visual and rapid detection of cow milk adulteration

A novel closed-tube loop-mediated isothermal amplification (LAMP) was developed to detect goat milk adulterated with cow milk. The reaction results of the LAMP assay developed, identified using both visual observation and agarose gel electrophoresis, were completely consistent, indicating that visual observation was suitable and efficient to judge the final LAMP results. The specificity of LAMP assay was evaluated using DNA of related species namely goat, pork, chicken, duck, dog, which revealed only a specific amplification of cow. The sensitivity of LAMP assay was as low as 10 fg. The limit of detection (LOD) of LAMP assay reached 0.01%, 10 times higher than PCR assay. In addition, this assay successfully amplified and identified 0.01% of cow commercial milk products in goat milk. Therefore, our results provided a simple, specific, sensitive, rapid, and cost-effective method to detect goat milk adulteration.

Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria.

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

Optimization of a Loop-Mediated Isothermal Amplification Assay as a Point-of-Care Tool for the Detection of Wuchereria bancrofti in Human Blood in Tana River Delta, Kenya.

Introduction Accurate detection of filarial parasites in humans and vectors is essential for the implementation and evaluation of Global and National Programs to eliminate lymphatic filariasis. Immunological methods to detect infection are available; however, cross-reactivity issues have been reported in most of them. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity and can be used to detect the infections. Methods In this study, we evaluated loop-mediated isothermal amplification (LAMP) tests to amplify Wuchereria bancrofti DNA in patients' blood. The amplicons were tested by both pH-sensitive dyes for enhanced visual detection and agarose gel electrophoresis. A closed-tube LAMP assay was also evaluated. Cohen's Kappa statistics was used for statistical analysis of the assays. 125 patients consented for blood sampling which were used for clinical analysis of LAMP assays with the PCR method used as the “gold standard.” Results The sensitivity of the evaluated Wuchereria bancrofti LAMP was 92.3%, with a specificity of 97.3% and kappa statistics value of 0.84, which is in a strong agreement. Conclusion In this study, LAMP assays coupled with fluorescence dye detection have been found to be suitable for diagnosis and monitoring of Wuchereria bancrofti infections in the Kenyan population.

Development of a novel loop-mediated isothermal amplification assay for rapid detection of Mycobacterium leprae in clinical samples.

Sensitive and definitive diagnostic tests are required for timely treatment of leprosy and to control its transmission. In the present study, we report the development of loop-mediated isothermal amplification assay using six primers targeting the RLEP gene sequence uniquely present in Mycobacterium leprae. Tissue punch samples (n = 50) and slit aspirates (n = 50) from confirmed cases of leprosy (M. leprae positive by quantitative polymerase chain reaction), reporting at the Department of Dermatology, Safdarjung Hospital, New Delhi, were analyzed using newly developed closed tube loop-mediated isothermal amplification assay. The sensitivity and specificity; positive predictive value, negative predictive value and accuracy were calculated using MedCalc statistical software. The loop-mediated isothermal amplification assay specifically amplified M. leprae genomic DNA with an analytical sensitivity of 100 fg. About 47 Out of the 50 quantitative polymerase chain reactions confirmed M. leprae positive tissue samples, 47 were positive by loop-mediated isothermal amplification assay (sensitivity 94%; 95% confidence interval 83.5%-98.8%) while only 31/50 were positive by histopathology (sensitivity 62%; 95% confidence interval 47.2%-75.4%) . Using slit aspirate samples of these 50 patients, 42 were positive by both quantitative polymerase chain reaction and loop-mediated isothermal amplification assay (sensitivity 84%; 95% confidence interval 70.9%-92.8%) while only 23/50 (sensitivity 46%; 95% confidence interval 31.8%-60.7%) were positive by microscopy. In the present study, the leprosy patient cohort was not uniform, as it comprised a lower number of paucibacillary cases (22%) compared to multibacillary (78%) cases. Loop-mediated isothermal amplification assay established here provides a rapid and accurate diagnostic test for leprosy in terms of sensitivity and specificity. The assay is simple to perform in comparison with other molecular techniques (polymerase chain reaction/quantitative polymerase chain reaction) and has potential for field applicability.

Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening method for virulent Aeromonas species affecting cultured eels in China

Under intensive and high-density aquaculture conditions, the cultured eels are easily infected by a variety of virulent Aeromonas species, which leads to high mortality rates and economic losses. In this study, a visual closed-tube loop-mediated isothermal amplification (LAMP) assay was developed for targeting aerolysin gene of Aeromonas species affecting cultured eels. Firstly, two pairs of outer and inner primers were designed against a conserved region of the aer gene from different Aeromonas species. Secondly, the weakly buffered aer-LAMP assay was optimized under various Mg2+ concentrations, different amplification times and amplification temperatures followed by the visual evaluation of phenol red dye and SYTO-9. Thirdly, the detection limit of the aer-LAMP assay showed 16.8 copy/μl which was simultaneously evaluated by the color changes from red to yellow and the real-time fluorescence amplification curves. The specificity of the aer-LAMP assay showed no cross-reaction with other related species. In addition, 30 Aeromonas strains and 40 clinical tissues from eels were used to further validate the effectiveness of the aer-LAMP assay. All the results showed that the aer-LAMP assay provided a rapid, simple, and reliable method for detecting Aeromonas harboring the aerolysin gene, which would be helpful for the prevention and control of Aeromonas infection in cultured eels.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4copies/μL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood

Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) based methods over serological tests and PCR for rapid detection of microbial pathogens. Here, a rapid LAMP assay was developed to detect the hepatitis B virus (HBV) from DNA, and particularly, blood samples from infected patients using a commercially available master mix and portable real-time fluorometer. The final optimized fluorescence-based LAMP assay provided significant amplification time of less than 15 minutes compared with over 1 hour for PCR and an opened tube LAMP system described previously. Results indicated that fluorescence-based LAMP assay was more sensitive than PCR as a rapid, sensitive, efficient, and highly reliable approach for rapid detection of HBV.

Rapid detection of collagens using a closed-tube LAMP method

Identification methods of collagens and gelatins have been studied many years due to religious and food safety issues. Some researchers detected the collagen while others took up their study based on DNA at the first time. In this work, we used a closed-tube loop-mediated isothermal amplification (LAMP) technique to differentiate collagen and gelatin samples. DNA was extracted by DNeasy mericon Food Kit and was dissolved in 30 µl elution buffer, optimum concentration of Mg2+, deoxyribonucleoside triphosphates(dNTPs), betaine in LAMP reaction is 6.0 mmol/L, 2.0 mmol/L, and 0.8 mmol/L, respectively. After LAMP reaction, samples being detected changed their initial color to green, others’ were colorless or brown slightly. The research offered a simple, fast detection technique to differentiate collagen and gelatin samples derived from porcine, bovine and channel catfish (Ictalurus punctatus) , the collagens’ species can be determined by color variation in reaction tubes within two hour.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

BackgroundLeishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application.MethodsWe have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL).ResultsThe assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse.ConclusionsThe study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis.

LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.

A Closed Tube Loop-Mediated Isothermal Amplification Assay for Identification of Brucella Species in Bull Semen

A closed tube loop-mediated isothermal amplification (LAMP) assay targeting omp25 gene was performed for detection of Brucella sp in semen from infected bulls. The assay was achieved through the use of reaction master-mix for performing LAMP reactions. The reaction master-mix contained all reaction components at their optimized concentration including Bst DNA polymerase and hydroxynaphthol blue (HNB) dye. The use of master-mix avoided opening of tubes after initial denaturation step required for adding Bst DNA polymerase. The HNB dye allowed visual monitoring of positive and negative LAMP reactions through naked eyes and hence circumvented need of opening reaction tubes for electrophoretic analysis of LAMP products. The use of master-mix simultaneously minimized contamination of laboratories, saved time and simplified reaction set up. The assay was performed in a simple water bath making it cost-effective. It was found specific to Brucella spp. only and could detect as few as 10 Brucella colony forming units in experimentally contaminated semen and 10 fg DNA (nearly 3 genome copies of Brucella) in pure culture. This sensitivity was found equivalent to that of TaqMan probe- based real-time PCR and 10 times higher than conventional PCR. Overall, the optimized LAMP assay was rapid, simple in operation, result oriented, highly sensitive and inexpensive. This could be used as a diagnostic test for detection of Brucella in semen at breeding bull stations in developing countries where laboratories are generally poorly equipped.

A Closed-Tube Detection of Loop-Mediated Isothermal Amplification (LAMP) Products Using a Wax-Sealed Fluorescent Intercalator

LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.