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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"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.
"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.
The Veterinary quarterly 07/2014; DOI:10.1080/01652176.2014.966172 · 0.72 Impact Factor
"Use of LAMP significantly reduced the processing time of sample and final outcome . Similarly, LAMP assay based on conserved region of “N” gene of PPR virus has been documented for rapid and specific detection of PPR virus from clinical samples. "
[Show abstract][Hide abstract] ABSTRACT: Irrespective of aetiology, infectious respiratory diseases of sheep and goats contribute to 5.6 percent of the total diseases of small ruminants. These infectious respiratory disorders are divided into two groups: the diseases of upper respiratory tract, namely, nasal myiasis and enzootic nasal tumors, and diseases of lower respiratory tract, namely, peste des petits ruminants (PPR), parainfluenza, Pasteurellosis, Ovine progressive pneumonia, mycoplasmosis, caprine arthritis encephalitis virus, caseous lymphadenitis, verminous pneumonia, and many others. Depending upon aetiology, many of them are acute and fatal in nature. Early, rapid, and specific diagnosis of such diseases holds great importance to reduce the losses. The advanced enzyme-linked immunosorbent assays (ELISAs) for the detection of antigen as well as antibodies directly from the samples and molecular diagnostic assays along with microsatellites comprehensively assist in diagnosis as well as treatment and epidemiological studies. The present review discusses the advancements made in the diagnosis of common infectious respiratory diseases of sheep and goats. It would update the knowledge and help in adapting and implementing appropriate, timely, and confirmatory diagnostic procedures. Moreover, it would assist in designing appropriate prevention protocols and devising suitable control strategies to overcome respiratory diseases and alleviate the economic losses.
Veterinary Medicine International 06/2014; Volume 2014 Special Issue(Article ID 508304):16 pages. DOI:10.1155/2014/508304