Introduction

Besides, the development of effective chemotherapies, infectious diseases continue to affect millions of lives around the world, especially in developing countries. Diagnosis of disease is important for adoption of proper therapeutic and prophylactic measures. There has been great revolution in the field of disease diagnosis over time. Earlier the basis for diagnosis was isolation and identification of the etiological agent from clinical specimens. Later, serological tests came up as diagnostic tools, which reduced the time for diagnosis. With the evolution of molecular techniques, the time needed for arriving at final diagnosis was cut down to hours from days (Dhama et al., 2014).

In the past few decades, several molecular methods have been developed to overcome the shortcomings of the classical diagnostics methods, especially the in-vitro amplification of a pathogen specific nucleic acid sequence. Such approaches may allow rapid diagnosis with a degree of sensitivity and specificity comparable to or even better than that of classical methods. However, “LAMP” which stands for loop-mediated isothermal amplification shows great potential for field use as it is simple and field-amenable. This technique was developed by Notomi and co-workers in 2000. The detection and enumeration of microorganisms in food are an essential part of any quality control or food safety plan. Traditional methods of detecting food borne pathogenic bacteria are often time-consuming, followed by isolation, biochemical and/or serological identification, and in some cases, sub specific characterization. Advances in technology have made detection and identification faster, more sensitive, more specific, and more convenient than traditional assays. LAMP has attracted a lot of attention as a potentially rapid, accurate, and cost-effective novel nucleic acid amplification method (Qin et al., 2016).

LAMP with its amenable nature used to detect a viruses, fungi, bacteria and parasites and in most of the cases it surpasses polymerase chain reaction. The challenges faced by the available technologies are mostly the cost of the kits and their use as standalone technologies, and hence LAMP holds a promise of being affordable and viable option. In this article, we have reviewed about the importance and applications of LAMP in field for rapid diagnosis of food borne and zoonotic pathogens.

Principle of LAMP

LAMP is a one-step amplification reaction that amplifies a target DNA sequence with high sensitivity and specificity under isothermal conditions. The mechanism of the LAMP reaction can be explained in three steps, an initial step, a cycling amplification step, and an elongation step. LAMP employs a DNA polymerase with strand-displacement activity, along with two inner primers (FIP, BIP) and two outer primers (F3, B3) which recognize six separate regions within a target DNA (Notomi et al., 2000). Fig.1 shows schematic representation of loop-mediated isothermal amplification (LAMP) primers.

LAMP Reaction

LAMP reaction mixture consists of DNA polymerase with high strand displacement activity i.e. Bst polymerase, four sets of primers- two inner primers and two outer primers, deoxy nucleotide triphosphates dNTPs, magnesium sulphate, betaine, buffer for enzyme, and template DNA.

Usually reaction is carried out at 60-65℃ for one hour. The target sequence consists of six distinct regions namely F3, F2, F1, B1c, B2c, and B3c in order from 5’ to 3’ end. Two outer primers are F3 and B3. Inner primers contain the sequence of both sense and antisense strands. FIP contains F1_C and F2. Similarly BIP contains B1_C and B2. Inner primers are added in higher concentration as compared to outer primers. Reaction gets initiated by binding of FIP to complementary sequence in target DNA, which is followed by strand displacement synthesis by outer primer F3. This produces FIP linked complementary strand, with loop at one end. BIP will bind on it, followed by B3. Thus, results in the production of a dumb bell shaped structure with loops at both ends. By self-primed synthesis, it produces stem loop structure, which acts as starting material for cyclic amplification step. Subsequently FIP binds to it and initiates synthesis by strand displacement producing stem-loop DNA with inverted copy of target sequence at stem and loop involving BIP at other end. On this structure, BIP binds and produces stem loop structure with twice long stem. This process continues. Final product of reaction is a mixture of stem loop DNAs of varying lengths and cauliflower like structures with multiple loops (4). Loop primers will bind to loop regions that are not bound by inner primers (Nagamine et al., 2004). Fig. 2 shows flow diagram of loop-mediated isothermal amplification (LAMP) for detection of Salmonella spp.

Detection of Amplified Products

A number of methods are available that can be used for detection of products in LAMP method. Amplified products can be directly observed by the gel electrophoresis, naked eye or using a UV trans-illuminator, intercalating dyes like SYBR green I stain, pitco green (Dukes et al., 2006; Tomlinnson and Boonham,2008) by-products from the reaction chemistry (Goto et al., 2009).

In positive cases, it will produce ladder like pattern due to the production of stem–loop structures with different stem lengths (Parida et al., 2008). The foremost advantage of LAMP in comparison with PCR is that the result of amplification can be interpreted without post amplification processing. Visual turbidity is a good indicator of positive reaction. Nucleic acids are amplified in large amount in LAMP reaction. This results in production of large excess of pyrophosphate ions, which will combine with magnesium ions resulting in production of white precipitate of magnesium pyrophosphate. This is responsible for turbidity in case of positive reaction (Mori et al., 2001). Increase in turbidity will be in direct proportion to the amount of nucleic acid, which can be measured real time using real time turbidimeter. Turbidity is measured as OD at 400nm in every 6 seconds. It is cheaper than real time PCR machine. For the formation of white precipitate, yield in microgram quantities is required. In LAMP, DNA amplified to more than 10µg, therefore, visual turbidity can be used as an indicator of positive reaction (Parida et al., 2008).

Polyethyleimine (PEI) can also be added to reaction tube post amplification for the detection of amplification. PEI forms insoluble complex with high molecular weight amplification product, but will not combine with low molecular weight oligonucleotides. Visually detectable clear coloured precipitate is formed on addition of PEI. But PEI cannot be added prior to reaction, as it will inhibit amplification (Mori et al., 2006).

Amplified products can also be visualised in presence of fluorescent intercalating dyes such as SYBR Green I, Calcein etc. On addition of SYBR Green I to the reaction tube post amplification, the colour changes from orange to green in case of positive reaction. Fluorescence can be detected visually using hand held UV torch (wavelength 365nm). Calcein is a fluorescent metal ion indicator. It can be added to tubes prior to reaction. Calcein quenches the manganese ions. Before reaction, solution appears orange in colour. As the reaction proceeds, manganese ions are released from Calcein and it will combine with pyrophosphate ions, thus increasing fluorescence of Calcein. Increased fluorescence can be detected visually as well as by ultraviolet light (Tomita et al., 2008). Colorimetric detection is also possible by addition of 120µM Hydroxynaphthol blue (HNB) to the reaction mix before amplification. HNB is a metal ion indicator. The colour of HNB changes depending on pH of the solution. Positive reaction is indicated by change in colour from violet to sky blue (Goto et al., 2009.).

Design of Primer

A set of two inner and two outer primers are required for LAMP. All four primers are used in the initial steps of the reaction, but in the later cycling steps only the inner primers are used for strand displacement synthesis. The outer primers are known as F3 and B3 while the inner primers are forward inner primer (FIB) and backward inner primer (BIP). Both FIP and BIP contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA, one for priming in the first stage and the other for self-priming in later stages. By using an additional set of two loop primers, forward loop primer (LF) and backward loop primer (LB), the LAMP reaction time can be further reduced. The size and sequence of the primers were chosen so that their melting temperature (T_m) is between 60-65 °C, the optimal temperature for Bst polymerase. The F1c and B1c Tm values should be a little higher than those of F2 and B2 to form the looped out structure. The Tm values of the outer primers F3 and B3 have to be lower than those of F2 and B2 to assure that the inner primers start synthesis earlier than the outer primers. Additionally, the concentrations of the inner primers are higher than the concentrations of the outer primers (Notomi et al., 2000).

Furthermore, it is critical for LAMP to form a stem-loop DNA from a dumbbell structure. Various sizes of loop between F2c and F1c and between B2c and B1c were examined and best results are given when loops of 40 nucleotides (40nt) or longer are used. The size of target DNA is an important factor that LAMP efficiency depends on, because the rate limiting step for amplification is strand displacement DNA synthesis. Various target sizes were tested and the best results were obtained with 130-200 bp DNAs (Dhama et al., 2014).

The certain characters to be considered while designing a primer for LAMP are-

1. Inner primers should not have AT rich sequence at both the ends.
2. GC content should be about 50-60%.
3. In case of GC rich sequence the melting temperature (Tm) should be 60-65^oC and for AT rich sequence it should be within 55-60 ^o C.
4. Designed primer should not have any secondary loop structure formation.
5. Distance between 5’ end of F2 and B2 should be 120-180bp and that of F2 and F3 is 0-20bp. The same can be considered for B2 and B3 also.
6. The distance for loop forming regions (5’ of F2 to 3’ of F1, 5’ of B2 to 3’ of B1) should be 40-60bp.

These primers for LAMP can be designed using online primer design software Primer- Explorer, http://www.Netlaboratory.com. (Zhang Tie et.al, 2012)

Table 1: Comparison of LAMP and PCR (Sahara, 2014)

Factors Affect the Efficiency of LAMP

Several factors affect the efficiency of LAMP reaction such as-

1. The efficiency of LAMP depends on size of the target DNA because one rate limiting step for amplification in this method is strand displacement DNA synthesis.
2. Hybridization of the four primers to the target DNA in the initial step is critical for the efficiency of LAMP.
3. The formation of stem-loop DNA from a dumb-bell structure is critical for LAMP cycling.
4. The size of the target DNA should be set to less than 300 bp.
5. Selection of appropriate DNA polymerase is also critical for LAMP efficiency.
6. Chemicals destabilizing the DNA helix have been found to markedly elevate amplification efficiencies in LAMP.
7. It has been shown that LAMP exhibits less sensitivity to inhibitory substances present in biological samples than PCR. This robustness of LAMP against inhibitors can contribute to saving the time and cost required for sample processing steps (Notomi et al., 2000; Kaneko et al., 2007).

Advantages of LAMP

1. LAMP is both sensitive as well as specific when compared with other DNA detection methods like PCR.
2. Large quantities of a targeted sequence (10⁹ -10¹⁰copies) are produced in less than an hour increasing sensitivity and the specificity for a positive reaction (Noren et al., 2011; Khan et al., 2012)
3. LAMP can be called as an equipment free technique because it does not require costly equipments and only water bath which is commonly available can be used. Hence, under field condition, the technique is suitable.
4. Requirement of fewer operation steps compared to other molecular detection methods. Hence detection of pathogen is more rapid.
5. Unprocessed or partly processed samples can be used as template and hence it is a robust technique. LAMP works at a constant temperature. No need of post amplification processing. Results can be seen directly by adding SYBR green, HNB or Calcein so electrophoresis is not needed which also reduces the time (Njiru, 2012).
6. Easy standardization among different laboratories.

Limitations

1. LAMP product is so firm that it is not degraded easily and chance of carry over contamination exist (Bai et al., 2011).
2. Use of impure DNA or partially purified DNA as template does not facilitate amplification (Degno et at., 2008)
3. LAMP is affected by amplification time. It was found that the least time taken for the amplification of LAMP varies from 60–120 min and negative control shows amplification at 180 min (Francois et al., 2011).

Applications of LAMP

1. Rapid Diagnosis of Zoonotic Disease

TB-LAMP is a new manual TB detection method based on the novel loop-mediated isothermal amplification (LAMP) platform from Eiken Chemical Co. in Japan. TB-LAMP has several features that makes it attractive as a diagnostics platform for resource-poor settings: it is fast (15-40 min), isothermal (requiring only a heat block), robust to inhibitors and reaction conditions that usually adversely affect polymerase chain reaction (PCR) methods, and it generates a result that can be detected with the naked eye. Since 2005, FIND and Eiken have been collaborating to develop an assay for TB that could be implemented in place of microscopy to improve the accuracy of TB detection at microscopy centres and similar laboratories (WHO, 2013).

Leptospirosis is considered to be the most widespread emerging zoonotic disease worldwide. Chen et al., (2015) developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations. Valladares and Rojo-Vázquez (2016) developed a LAMP assay to improve the diagnosis of Fasciola spp. in the faeces of sheep. They described that LAMP assay could be a good alternative to conventional diagnostic methods to detect F. hepatica in faeces since it solves the drawbacks of the standard PCR. The rickettsial bacterium Ehrlichia ruminantium causing heartwater disease not only responsible for high economic losses in endemic countries, but is also suggested to be a potential emerging zoonosis. Since, the LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of pathogen’s presence in human cases in sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland (Kundapur and Nema, 2016). Thus, LAMP is providing a great platform for quick and accurate identification of different pathogens in medical as well as veterinary field.

2. Molecular Diagnosis

The loop-mediated isothermal amplification (LAMP) technique has the potential to revolutionize molecular biology because it allows DNA amplification under isothermal conditions and is highly compatible with point-of-care analysis. LAMP is widely being studied for detecting infectious diseases such as tuberculosis, malaria, and sleeping sickness. In developing regions, it has yet to be extensively validated for other common pathogen. LAMP has been observed to be less sensitive than PCR to inhibitors in complex samples such as blood, likely due to use of a different DNA polymerase (typically Bst DNA polymerase rather than Taq polymerase as in PCR). Several reports describe successful detection of pathogens from minimally processed samples such as heat-treated blood or in presence of clinical sample matrices (Curtis et al., 2008).This feature of LAMP may be useful in low-resource or field settings where a conventional DNA or RNA extraction prior to diagnostic testing may be impractical.

Maeda et al., 2009 successfully developed the LAMP assay for the diagnosis of chronic myeloproliferative neoplasms. The myeloproliferative disorders are a group of haematological conditions at the level of the multipotent haematopoietic stem cell leading to increased production in one or more blood cell types. In an interesting study, Yoneda et al. (2014) developed a RT-LAMP assay using cytokeratin 19 as a target gene for the detection of free cancer cells in peritoneal lavage and assessed the clinical significance of the molecular diagnosis by survival analysis and frequency of recurrence with a median follow-up period of 39 months.

3. Detection of Genetically Modified Organisms (GMO)

The loop-mediated isothermal amplification (LAMP) assay indicates a potential and valuable means for genetically modified organism (GMO) detection especially for its rapidity, simplicity, and low cost. There is an increased trend of introduction of genetically modified crops in the field of agriculture that created a necessity for the development of rapid, economic and effective on-site detection methods. The risk of the introduction of GM product into the environment or food chain has become an important issue of bio ecology and biosafety concern. Therefore, it is an important practical significance to detect GM crops from different trading sites. Even though the PCR technique is available but it has its own drawbacks. The LAMP assay is an alternate and can be performed on site. The cry1Ab gene is a foreign gene which encodes Bt insecticidal Cry1Ab protein and was transferred into genomic DNA of plants to acquire insect resistance. Loop-mediated isothermal amplification (LAMP) assay with high specificity and rapidity under isothermal conditions was developed for detecting cry1Ab gene in transgenic rice. The LAMP assay was successfully employed for the detection of different GM event and was proved to be sensitive than the routine PCR (Abdulmawjood et al., 2014)

4. Rapid Identification of Food Adulterations

An easy, rapid and sensitive method of detection of the presence of meat species in raw or processed foods is important from cultural, religious, health and commercial perspectives. To address the issue of authenticity of imported Ostrich meat, that is found adulterated with either beef or other less-expensive meat or with wild Ostrich species in and around Europe, (Beversdorf et al., 2015) developed a LAMP assay and designed primers for conserved region of cytochrome b of mitochondrial DNA. The total DNA was isolated from 27 Ostrich samples procured from local market and through online purchase. Concurrently, they also procured reference DNA from cow, pig, sheep, goat, turkey, chicken, dog, cat, horse, and deer for identification. The LAMP assay was performed without much change except a melting curve analysis at the end of the assay and the reading of results was done on a real-time fluorometer. The assay showed a specificity of 100% with the investigated samples. The assay could detect 1 g of ostrich meat in 10 kg of meat product. Similarly, they showed the robustness of the LAMP assay by performing LAMP assay on DNA extracted from heat-treated as well as from fried meat with oil and spices gave very good results which makes a suitable assay in the investigation of food samples in restaurants or even of canned meat samples. This was the only report which addressed the issue of food adulteration with the help of LAMP assay.

5. Detection of Food Allergens

Zhang et al., 2013, establish a detection method of allergen soybean from food by loop-mediated isothermal amplification assay (LAMP). The soybean could be rapidly and specifically detected by LAMP method within 40 min at constant temperature of 63°C. The method was specific for soybean, and the limit of detection was determined as 0.01% (w/w) soybean powder. Celery (Apium graveolens) is a widely used ingredient in seasonings, sauces, bouillons, and instant meals should be labelled according to Directive 2003/89/EC due to their allergenic potential. Zahradnik et al. (2014) selected celery-specific mannitol dehydrogenase and developed LAMP assay. The celery consumption produced severe allergic reactions in some individuals in Europe especially in central Europe, mainly France, Switzerland, and Germany. The allergic complications include digestive disorders, respiratory distress, and skin reactions. But based on the literature recommendation, the performance of the LAMP assay developed was found to be equal or superior to the best available PCR assay for the detection of celery in food products (Hua et al., 2014).

6. Detection of Pesticides

Over the years, the widespread use of pesticides has had several benefits and also caused many problems. Hua et al. (2014) developed iLAMP which is a rapid, sensitive, and economical method for detecting Organophosphorus (OP) pesticides and their residues in food and the environment and in future can be used for the detection of other small molecules. OP pesticides are considered as hazardous substances because of their toxicity to nonpests and bioaccumulation and biological magnification in the environment. The group used four phage-borne peptide mimotopes with specific affinities to a monoclonal antibody (mAb) against OP pesticides as a secondary reagent and came out with an iLAMP with higher sensitivity than ELISA. The group evaluated 23 OP pesticides. One assay was used to screen eight OP pesticides with LOD between 2 and 128 ng/ml. This was the first report were LAMP assay was successfully used to detect small molecules like pesticides.

Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient So, Loop-mediated isothermal amplification (LAMP) an efficient and simple method with high specificity was developed based on drug resistance (Liu, 2012). The drug resistant micro-organisms are responsible for prolonged illnesses and high mortality rate. The LAMP assay is now successfully addressing the serious issue like detection of drug resistance. Liu et al. (2012) successfully developed LAMP assay for the detection of New Delhi Metallolactamase 1 (NDM-1) carrying isolates which confer resistance to carbapenem and proved that LAMP assay was highly sensitive technique for the rapid detection of blaNDM-1 which confer the resistance to carbapenem. This assay was successfully tested and conformed on pure culture, sputum, urine and faecal samples.

Similarly, Qi et al., (2012) developed LAMP assay for the detection of blaNDM-1 and another well-known antibiotic resistant gene cfr (chloramphenicol-florfenicol resistance). The cfr rRNA methyltransferase known to confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics.

7. Sex Determination in Endangered Species

The LAMP assay technique has been playing vital role in rapid and non-invasive identification of sex and species of this critically endangered species. To conserve an endangered species identification of species and its sex are the prime factors, such identification is not easy in all organisms and one such example is an endangered Formosa landlocked salmon (Oncorhynchus masou formosanus). As morphological differences are very minimal in salmon. Hsu, et al. (2011) designed the LAMP assay for the same. This is the first report where LAMP was successfully developed. LAMP primers were designed for growth hormone GH 1 gene for species identification and the male-specific marker (OtY2m; GU181208) for sex identification.

Future Prospectus of LAMP

Emergence of LAMP has focused on the grey areas in molecular diagnostics and has made it affordable to low resourced laboratories in developing countries like India. This unique quality of LAMP has led to its adoption in diagnosis of many infectious diseases. Health regulators in most countries, as well as international health bodies, such as the World Health Organization (WHO) and other national and international health regulatory bodies, recommends LAMP for diagnosis of some infectious diseases of medical and veterinary importance. LAMP diagnostic kits are currently available for clinical diagnosis of some infectious diseases, which reflects the quality and usefulness of the technique. Furthermore the importance of nucleic acid amplification in various fields of science, such as forensic investigation, food science technology, genetics and clinical diagnosis, underscores the need to develop a robust technique that will bridge the gaps in molecular diagnostics in both medical and veterinary science.

Acknowledgment

This review is based on research work from the project ‘Detection of food borne pathogens by LAMP (Loop Mediated Isothermal Amplification) Technology supported by grants from Indian Council of Agricultural Research, Govt. of India, New Delhi-110 001 under the research Project “All India Co-ordinated Research Project on Post Harvest Engineering and Technology” implemented at Bombay Veterinary College, Parel, Mumbai-400 012

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