Diagnosis is an important part in case of animal husbandry as treatment of a disease depends on it. Advancement in molecular biology has generated various sophisticated tools like Polymerase Chain Reaction (PCR), its versions along with pen-side diagnostic techniques. Every diagnostic test however has both advantages and disadvantages; PCR is not an exception to this statement. To ease the odds faced by PCR several non-PCR techniques which can amplify DNA at a constant temperature has become the need of hour, thus generating a variety of isothermal amplification techniques including Nucleic Acid Sequence-Based Amplification (NASBA) along with Self-Sustained Sequence Replication (3SR) and Strand Displacement Amplification (SDA) and Loop mediated isothermal amplification (LAMP) test. LAMP stands out to be a good and effective diagnostic test for empowering in developing countries as it does not require sophisticated equipments and skilled personnel and proves to be cost-effective. Performance of LAMP mainly relies on crafting of six primers (including 2 loop primers) ultimately accelerating the reaction. LAMP amplifies DNA in the process pyrophosphates are formed causing turbidity that facilitates visualisation in a more effective way than PCR. The Bst and Bsm polymerase are the required enzymes for LAMP that does not possess 5'-3' exonuclease activity. Results can be visualized by adding DNA binding dye, SYBR green. LAMP is more stable than PCR and real-time PCR. Non-involvement of template DNA preparation and ability to generate 10(9) copies of DNA are added benefits that make it more effective than NASBA or 3SR and SDA. Thus, it fetches researcher's interest in developing various versions of LAMP viz., its combination with lateral flow assay or micro LAMP and more recently lyophilized and electric (e) LAMP. Availability of ready to use LAMP kits has helped diagnosis of almost all pathogens. LAMP associated technologies however needs to be developed as a part of LAMP platform rather than developing them as separate entities. This review deals with all these salient features of this newly developed tool that has enlightened the world of diagnosis.
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"Recently, a rapid and simplified molecular technique, the loopmediated isothermal amplification (LAMP) has been shown to be an effective tool in detection of human pathogenic infectious agents (Notomi et al., 2000; Mori et al., 2012; Dhama et al., 2014). The technique has been applied in the detection of Leishmania using purified DNA extracted from patient's materials (Takagi et al., 2009; Adams et al., 2010; Khan et al., 2012) or swab boiled samples from CL model mice (Direct Boil-LAMP method; Mikita et al., 2014). "
[Show abstract][Hide abstract] ABSTRACT: Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64°C for 60min using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per μl. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas.
"Background Loop mediated isothermal amplification assay (LAMP) has made diagnosis simple at field level because of its swiftness, sensitivity, specificity and adaptability. LAMP has been developed for many pathogens with good success as a diagnostic tool compared with other molecular techniques . LAMP assay has numerous advantages like working at constant temperature, no need of sophisticated instruments, no need of post amplification modification, good sensitivity and specificity hence it could easily be used as a diagnostic assay at field level. "
[Show abstract][Hide abstract] ABSTRACT: Loop mediated isothermal amplification (LAMP) assay, a promising diagnostic test, has been developed for detection of different pathogens of human as well as animals. Various positive points support its use as a field level test but the major problem is product cross contamination leading to false positive results. Different methods were adopted by various researchers to control this false positive amplification due to cross contamination but all have their own advantages and disadvantages. A new closed tube LAMP assay based on agar dye capsule was developed in the present study and this technique has some advantages over the other closed tube technique.Agar at the concentration of 1.5% was used to sandwich SYBR green dye I with the aid of intradermal syringe. This agar dye capsule was placed over the LAMP reaction mixture before it was amplified.To eliminate the hazardous nature of Ultra Violet (UV) light during result visualization of LAMP products, the present study demonstrates the use of Light Emitting Diode (LED) lights for result visualization.LAMP was carried out for Brucella species detection using this modified techniques yielding good results without any cross contamination and LED showed similar fluorescence compared to UV. Method
Closed Tube LAMP technique.
"instruments and its results can be viewed directly with no post-amplification protocols. It has higher sensitivity and specificity compared to PCR and hence can be used as a valuable diagnostic tool at field level (Notomi et al. 2000; Nagamine et al. 2002; Dhama et al. 2014). To our knowledge , there are only seven reports regarding detection of Brucella spp. "
[Show abstract][Hide abstract] ABSTRACT: Abstract Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen needs to be diagnosed at an early stage. Loop mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with PCR. Results: The developed LAMP assay works at 61°C for 60 min and the detection limit was observed to be 100 fold more than the conventional polymerase chain reaction (PCR) that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with RBPT and STAT. SYBR green dye I was used to visualize the result with naked eye. Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.
No preview · Article · Jul 2014 · The Veterinary quarterly