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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	��j�bjbj����	4�ΐΐ�!�������&&i i i i i ����} } } } �#,} J���%�Y&Y&Y&Y&H'H'H'�����������:�i H'H'H'H'H'�i i Y&Y&���N�N�NH'Z%i Y&i Y&��p�NH'��N�N�#�tK�Y&�����6��!��} �L���:m��0J�эz��tN|��tK�K�f��i ���H'H'�NH'H'H'H'H'���NH'H'H'J�H'H'H'H'����������������������������������������������������������������������H'H'H'H'H'H'H'H'H'&	/:	Loop mediated isothermal amplification system (LAMP): a comprehensive review with special reference to veterinary medicine
G. Venkatesan*, Anand Kushwaha, Amit Kumar, G. Poulinlu, Monu Karki and P. Sasikumar

Pox virus laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteswar 263 138, Nainital District, Uttarakhand, India.










*Corresponding author
G Venkatesan
Ph: +91 5942-286346
Fax: +91 5942-286347
Email address: HYPERLINK "mailto:gnanamvirol@gmail.com"gnanamvirol@gmail.com

ABSTRACT 
Loop mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, sensitive and specific nucleic acid amplification assay at a constant temperature in water bath/heat block by using Bst DNA polymerase. There is a need for an inexpensive, robust diagnostic technique as an alternative to PCR and qPCR assays. The LAMP assay is found to be 10-100 fold more sensitive than the PCR. This technique uses four specially designed primers capable of recognizing six different regions in the target DNA so that the specificity is extremely high. There are several detections methods that are available for one-pot closed tube amplification reactions to minimize/control the carryover contamination during the opening of amplified tubes. LAMP technique is employed in the clinical diagnosis of pathogens including viruses and other applications namely single nucleotide polymorphism (SNP) typing, inspection of meat adulteration, and detection of GMOs, sex determination, detection of pesticides, tumor detection and quantification of template DNA. As a closed-tube LAMP method, the LAMP can be used as a field diagnostic tool in low resource settings where the diagnostic testing may be impractical. This review emphasis on the principle of LAMP, methods of detection, modifications of LAMP, advantages over routine molecular diagnostics and its applications in veterinary medicine.
Keywords: Isothermal amplification; LAMP; monitoring methods; visual detection; diagnosis applications: 







Introduction 
Nucleic acid amplification is most commonly used technique for diagnosis, in research, forensics, medicine and agriculture. Polymerase chain reaction (PCR) is the most widely used nucleic acid amplification tool for the diagnosis of several infectious and non-infectious diseases of animals and humans. But, it requires high expensive instrumentation for amplification and analysis which also needs the technical expertise. There are different isothermal nucleic acid amplification methods have been invented to overcome the disadvantages of PCR and related assays that require high cost equipment and expertise. These isothermal amplification systems used for amplification of DNA, including nucleic acid sequence based amplification (NASBA) [1], strand displacement amplification (SDA) [2], self-sustained sequence replication reaction (3SR) [3], transcription-based amplification system (TAS) [4], rolling circle replication (RCR) [5], helicase-dependent amplification (HDA) [6], single primer isothermal amplification (SPIA) [7], cross-priming amplification [8] and loop-mediated isothermal amplification (LAMP) [9].  Among these amplification methods, like RCR, LAMP and CPA can be amplified product at a constant temperature. LAMP assay is an alternative to PCR based methods for rapid and early detection of most of the infectious diseases in the field diagnostic assay [10].  
The LAMP is a highly specific and sensitive reaction [9,11]. LAMP assay can be amplified at a constant temperature by water bath/heat block by using Bst polymerase large fragment and it is used for development of POCT (point of care testing) in a field [12]. The LAMP assay can also be used to quantify the amount of amplified DNA [13, 14,15]. The reactions are tolerant against inhibiting substances in samples [16]. The LAMP assay amplifies few copies of non-denatured DNA into billion copies (up to 109) within an hour of reaction time [12,9]. The LAMP assay was found to be 10-100 fold more sensitive than that of conventional PCR and detection limit was found to be in the range of 0.01-10 pfu for viruses [17]. 
Principle of LAMP assay
Bst DNA polymerase has high strand displacement and polymerase activities at a constant temperature so that the assay can be performed at a constant temperature. The assay uses four specially designed primers capable of recognizing six different regions in the target DNA so that the specificity is extremely high [9]. Unless all the target regions are available, the amplification will not proceed. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP reaction and followed by a strand-displacement DNA synthesis primed by an outer primer releases a single stranded DNA. This serves as a template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop structure. In subsequent LAMP cycling, one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand (The Eiken Chemical Company, Japan).
LAMP Primer Design and assay set up
The proper selection of the target gene and the design of primers are critical and essential for successful amplification of target DNA by LAMP assay. Use of primer designing software �Primer explorer� exclusively for the LAMP assay is recommended ( HYPERLINK "http://primerexplorer.jp/e/" http://primerexplorer.jp/e/) Another linux based software LAVA is also available [18]. Primer explorer support page indicates other critical parameters to be considered while designing the primers. The additional sets of loop primers can be designed, which can further speed up the reaction rate and the amplification process. The assay is usually setup in 25�l reaction, containing 40-50 pmol each of the primers FIP and BIP, 5-10 pmol each of the outer primers F3 and B3, 20-25 pmol each of loop primers FLP and BLP, 2x reaction mixture of reaction buffer having 20 mM Tris-HCl pH8.8, 10 mM (NH4)2SO4, 10 mM KCl, 0.8-1M betaine, 4-8 mM MgSO4, 1-3 mM dNTPs, 0.1% Tween 20, 8 units of the Bst DNA polymerase and 1-2 �l of DNA template. The assay can run at a single constant temperature ranges in 60-65�C in a water bath or heating block or a thermal cycler. For RNA template, one step reverse transcription Loop mediated isothermal amplification (RT-LAMP) technique used by using 1U of Avian myeloblastosis virus reverse transcriptase (AMV-RT) in above LAMP reaction mixture that synthesized cDNA from RNA template and is further amplified by using Bst DNA polymerase. RT- LAMP assay is very effective and time saving because the reaction can be completed in a sigle step at constant tepmerature [19,20].  At the end of the reaction, a denaturation step of 80�C can be used.

Factors to be considered during designing of LAMP primers
GC content should be 50-60 %.
Inner primers should not have AT rich regions at both the ends.
Primer should not have any secondary loop structure formation.
In case of AT rich sequence the melting temperature (Tm) should be 55-60�C and for GC rich sequence it should be within 60-65�C.
The distance between 5� end of F2 and B2 should be 120-180bp and for F2 and F3 is 0-20bp. The same factor considered for B2 and B3.
The distance for loop forming regions (5� of F2 to 3� of F1, 5� of B2 to 3� of B1) should be 40-60bp.
LAMP Vs PCR 
PCR based method requires high expensive instruments, skilled operators, and data analysis. PCR based molecular diagnostics also requires different methods for detection of amplified DNA products. PCR and qPCR have some disadvantages like the requirement of the expensive thermal cycler, time-consuming, computer, data analysis software, optics for �uorescence excitation and emission collection and contamination by analysis of PCR products [17]. Real-time PCR machines are more expensive so it is difficult to purchase in developing countries. Apart from this, they are difficult to adapt as a field diagnostic tool, especially in developing countries. The LAMP can detect up to femtogram level of DNA. There is no need for processing of DNA samples, and impurities would not hinder the reaction [16,21]. So, as a field diagnostic tool, there is a need for sensitive, inexpensive, robust diagnostic technique which is alternative to PCR and qPCR.





Detection methods in LAMP assays 
By turbidity or precipitates of magnesium pyrophosphates [22,13,9] and real-time monitoring by spectrophotometric analysis [17].
DNA-intercalating dye, like SYBR green and Picogreen [9,17,23] added after reaction or using a wax-dye capsule or agar plug method
Metal ion-binding �uorophore, such as calcein [24] and bycolor change using a metal ion-binding indicator dye such as hydroxy naphthol blue (HNB) [25] and acid chrome blue K (ACBK) [26].
Agarose gel analysis [17].
Sequence-specific visual detection of the LAMP using polyethylenimine (PEI) [17].

By turbidity or precipitates of magnesium pyrophosphates
                  The LAMP reaction produces large amount of pyrophosphate ions as a byproduct which interacts with magnesium ion of reaction buffer and forms a white precipitate of magnesium pyrophosphates in the reaction tube. The amount of DNA synthesizes directly correlates with the amount of pyrophosphates production. The LAMP assay is also detected by the real-time measurement of turbidity with the help of spectrophotometric analysis turbidimeter. Turbidity indicates is due to the higher amplification efficiency of LAMP assay [22,13,17].

A direct method using DNA-intercalating dye
	SYBR green is a fluorescent dsDNA intercalating dye added to the tube for end-point detection. The reaction is detected by a color change from orange (negative) to green (positive). The color change in the reaction tubes is visualized by the naked eye and or UV transilluminator. These are highly sensitive but more expensive and toxic. SYBR Green has an inhibitory effect on DNA amplification.  To prevent the inhibitory effect of SYBR green it can be added after the reaction is completed but it required opening of tubes at the end of reaction which leads to increased the risk of cross contamination [9,17,23]. Another intercalating dye Picogreen can be added in the tube  after reaction is completed and the color change of Picogreen from orange to yellow green indicated positive reaction [27]. EvaGreen used for real-time monitoring of LAMP reactions and it does not inhibit the reaction [28].
Agar dye capsule technique
                   Agar dye capsule technique is a closed-tube method consists of 1.5% agar and SYBR green I dye. The capsule should be placed over the reaction mixture prior to incubation. The reaction tubes are visualized under LED lights. Normally, SYBR Green dyes are visualized under UV lights but in this technique LED lights are used for visualization. This closed-tube LAMP method used for detection of Brucella�species (B. abortus�S99,�B. abortus�S19,�B. melitensis,�B. suis) [29].

Wax-dye capsule method 
Wax sealed SYBR green I dyes are used in the closed-tube reaction [30]. �The wax melted during amplification when exposed to a higher temperature and the products are detected by the released dye. This method used for visualization of closed-tube LAMP technique for field detection of�plasmodium vivax�infection [31].

Agarose gel analysis
               After the amplification, the LAMP products are detected by using agarose gel in the Tris-borate buffer, followed by and staining/stained with ethidium bromide and agarose gels are visualized under UV transilluminator. The amplified products are detected by post-amplified detection methods, so it requires the opening of the LAMP tube and it leads to a carryover contamination [17].
An indirect method using metal-ion indicator dyes
Colorimetric method is a type of indirect closed-tube detection method detects amplified products by using metal ion indicator dyes. Examples of metallic ion indicator dyes are Hydroxynaphthol blue (HNB) [25], calcein [24] and CuSO4 [32]. These dyes are added as pre-addition reagents with other lamp reagents.
Hydroxynaphthol blue (HNB) 
                     HNB is a type of synthetic colorimetric dye for magnesium ions. HNB widely used to differentiate positive and negative reactions [28] in a closed tube method as well as for post amplified detection.  The color change of HNB from violet to sky blue indicated positive reaction. For definitive naked eye endpoint visual detection, HNB is the simple and sensitive method [33].
Calcein
                    Calcein is a synthetic fluorescein dye used for both end-point and real-time detection of amplification [34]. Calcein produces fluorescence by complexes with magnesium ions. The sensitivity of calcein was 10 times lower than HNB because of manganese inhibits the reaction [25].
         c) Acid chrome blue K (ACBK)
               ACBK is a metal-ion indicator dye. In positive reactions, the color change from red to blue and visually detected by naked eye or UV light. The mechanism of ACBK is similar to that of HNB. ACBK LAMP was used for detecting Cauliflower Mosaic Virus 35S promoter [26].
          d) Malachite green (MG)
    Malachite green (MG) is a pre-addition non-fluorescent cationic dye used for simple visual detection of LAMP reactions by the naked eye. After the amplification, a color change to light blue (positive samples) and colorless (negative samples). This method does not require any special instruments like UV illuminator or lightbox for detection. Malachite green is a very cheap dye, does not require freeze storage and easy to prepare [35,36]. �Malachite green was used for end-point detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes [37].
Sequence-specific visual detection of the LAMP using Polyethylenimine (PEI)
                 This simple method detects LAMP products in a sequence-specific manner visually by adding polyethylenimine (PEI) to the LAMP reaction solution. PEI has a strong inhibitory effect on reaction, so it should be added after the reaction starts. PEI yields precipitate with a clear color and in a size that can be identified visually [38,17].







Carryover contamination in the LAMP
Because of high sensitivity of LAMP assay, it is very vulnerable for carry over cross contamination and gives false positive results due to re-amplification of highly concentrated amplified products. These re-amplification leads to contamination by aerosols, air, reagents, equipment,  and clothes [4,24]. This is called carryover contamination. Initially amplified products are visualized by post amplified detection methods and gel electrophoresis that requires the opening of reaction tubes and carryover contamination occurs [17]. There are several detections methods are available for one-pot closed tube amplification reactions to minimize/control the carryover contamination.

Closed-tube LAMP assay
To reduce the contamination, researchers developed closed-tube LAMP assay. In this method, indicator dyes are added together with other LAMP reagents before an amplification starts. This method cannot eliminate contamination if contamination occurs. Closed-tube detection methods use magnesium pyrophosphate precipitation [22,13], Malachite green [35], Wax-dye capsule method using SYBR green I [31,30], Hydroxynaphthol blue (HNB) [25], Calcein [24], Acid chrome blue K (ACBK) [26], Agar dye capsule technique [29], automated endpoint detection system�for LAMP [46]. Among these methods, only a few methods are eligible for ��ASSURED�� guidelines like magnesium pyrophosphate turbidity and precipitation, by use use of the metal-ion indicators like Hydroxynaphthol blue (HNB) and Calcein dyes (colorimetric method).
Principle of indirect detection/metal indicator dye method (colorimetric method)
              During DNA elongation, insoluble magnesium pyrophosphates are formed as a by-product of magnesium ions and pyrophosphate ions from MgSO4 and dNTPs in the reaction mixture respectively. The LAMP is a highly sensitive assay, so it can produce a high amount of DNA (400 �g/mL or 1039 copies of DNA) [24,17]. Because of its high amount of DNA, high amount pyrophosphates are produced [24]. The LAMP amplification detected by turbidity formation as a result of magnesium pyrophosphates salts [22], but it is very difficult to monitor for visual detection. The changes in the metallic ion concentrations are visually detected by using different methods [24]. Among four methods to detect metallic ion concentration (Flame analysis method, colorimetric method, electrode method and gravimetric method), colorimetric method may be applicable and reliable in the field for visual detection to avoid carry-over contamination as these are simple, economical and reliable to use in field conditions [24].
Colorimetric method/Metal indicator dye method
             Colorimetric method is a type of indirect detection method detects amplified products by using metal ion indicator dyes. As the target gene is amplified the color of LAMP solution will be change, which can be easily visualized by naked eyes and it does not required any extra detection instruments [42]. Examples of metallic ion indicator dyes are Hydroxy naphthol blue (HNB) [25], calcein [24] and CuSO4 [32]. These dyes are added as pre-addition reagents with other lamp reagents.  
Hydroxynaphthol blue (HNB)
                  HNB is a type of synthetic colorimetric dye for magnesium ions. HNB widely used to differentiate positive and negative reactions [28] in a closed tube method as well as for post amplified detection. Compared with the calcein and other methods, HNB is the best method [47] for field assay due to its easy, inexpensive and dependable interpretation of the result. There is no requirement for any types of equipment for endpoint detection. For definitive naked eye endpoint visual detection, HNB is the simple and sensitive method [33]. HNB never affect the detection limit of the reaction [47] and the detection limit was 2 copies/ Reaction [48]. HNB detection sensitivity was equal to SYBR Green [25] and higher than turbidity [49,25,50,51].

Mechanism of action: 
               HNB is commonly used for end point titration of Mg2+ at pH 10.0. HNB is a magnesium-chelating dye (violet) and the color change depends on Mg2+ and pH of the reaction mixture [25]. During LAMP reaction pyrophosphates produced that binds with magnesium ions and form a white precipitate of magnesium pyrophosphate, leads to free HNB (sky blue). The reaction is visually detected by a color change from violet (Negative) to sky blue (Positive).
Calcein
                Calcein is a synthetic fluorescein dye used for both end-point and real-time detection of amplification [34]. Calcein produces fluorescence by complexes with magnesium ions. The sensitivity of calcein was 10 times lower than HNB because manganese inhibits the reaction [25].
Mechanism of action
             During LAMP reactions, Calcein with Manganous chloride (Mn2+) added to the reaction system as a pre-addition dye. Manganous ions were provided for quenching the fluorescence of calcein. As DNA elongation proceeds, pyrophosphates produced that form complex  with manganous ions, which results in the emission of fluorescence from calcein. The free calcein combines with magnesium ions present in reaction buffer, which enhances the fluorescence of calcein or color change [24]. Complex of manganese pyrophosphates provides white turbidity to the reaction mixture, which forms white precipitates after centrifugation. The positive reactions are visually detected by a color change from orange to green/greenish yellow color by the naked eye or by UV light, positive samples excite bright green fluorescence.
Advancements or modifications in the LAMP system
Multiplex LAMP (mLAMP)
mLAMP is a reliable method for the detection of multiple target genes simultaneously. In this method, LAMP primer sets are specially designed for different serotypes or species and reaction carried in a single tube tune LAMP. mLAMP approach for detection of the dengue virus, Taenia solium, Taenia saginata, Taenia asiatica, Salmonella spp., Vibrio parahaemolyticus, Plasmodium berghei and Dirofilaria immitis [52].
Electric LAMP (eLAMP)
An electric LAMP is efficiently used to test putative LAMP primers on a target sequence. eLAMP can match primers with templates by using built in PERL regular expressions (exact matching) or via the tre-agrep library (approximate matching). eLAMP was used for specific amplification of staphylococcus aureus genomes and not amplify from other Staphylococcus species [53].


Micro LAMP
In this modified LAMP system, microfluidic chip is used for the quantification and detection of nucleic acid. It is also used for the real-time quantification of genes by using an optic fibre in the micro LAMP. The micro LAMP standardized for detecting Peseudorabies virus (PRV) and also for SARS, tuberculosis, and influenza A (H1N1) [54]. 
Lateral flow assay (LFA) in the LAMP
LAMP with chromatographic lateral flow dipstick (LFD) detects biotin-labelled amplified products that are hybridized with a fluorescein isothiocyanate (FITC)-labelled DNA probe  complexed with a gold-labelled anti-FITC antibody. This method does not require any special equipment. It provides highly specific, rapid, and simple visual detection method [55-57]. However, for LFD detection requires the opening of tubes, so it leads to contamination. LAMP employs an absorbent strip, containing an antibody specific to a target sequence. The absorbent strip binds to the LAMP product in a positive reaction. Loop mediated isothermal amplification combined with nucleic acid lateral flow assay used for the rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) and cyprinid herpes virus and Vibrio vulnificus [58]. 

Real-time turbidimeter in the LAMP
 LAMP reaction byproduct, pyrophosphates ions binds with magnesium ions and form precipitate of magnesium pyrophosphate that leads to turbidity. Changes in the turbidity of LAMP reaction mixture measured by real-time turbidimeter is used for the real-time monitoring and quantification of LAMP product. This promotes the user-friendliness and ease of LAMP [13].
Loop mediated isothermal amplification with lateral flow biosensor (LAMP-LFB)
LAMP-LFB is used for specific, visual, and multiplex detection of the nucleic acid sequence. It is used to detect various target sequences by re-designing the specific LAMP primers and also detect a single target. Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensors were used for detection of Enterococcus faecalis and Staphylococcus aureus [59].
In-disc LAMP (iD-LAMP)
iD-LAMP is a device consists of micro-reactors embedded onto compact discs and it is used for real-time detection of targeted nucleic acid [60]. This method requires an only a smaller amount of template and carried out in the oven at 65�C for amplification. In disc LAMP assay used for detection of pathogenic Salmonella spp. and identification of bovine meat in meat samples by using the principle of the in-disc LAMP (iD-LAMP) and quantitative optical read- out by a disc drive [52].
Fluorescence of Loop Primer Upon Self Dequenching-LAMP (FLOS-LAMP) 
In this method, a labelled loop probe quenched in its unbound state fluoresces only when bound to its target. It is used for the sequence-specific detection of LAMP amplicons. The FLOS-LAMP concept was validated for rapid detection of the human pathogen, Varicella-zoster virus, from clinical samples [61].

Detection of the LAMP by portable multi-channel turbidity system 

The portable multi-channel turbidity system includes photoelectric detection unit, a temperature control unit, power management unit, turbidity calibration unit, a communication unit, human machine unit and ARM-based microcontroller. It is developed for real-time monitoring of LAMP reaction and it is characteristics by small size, low cost, high-efficiency, excellent stability and high uniformity. Legionella bacteria (LEG) and H7 subtype virus (H7) were successively detected�designed and developed a portable multi-channel turbidity system [62]. �
Visual detection of the LAMP by using gold nanoparticles 

Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs) and engineered to signal the LAMP reaction. The loop primers of the LAMP were biotinylated and then ca nproduce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex and this leads to a color change of the reaction from red to blue. The LAMP by using gold nanoparticles was used for targeting the species-specific�tlh�gene of�Vibrio parahaemolyticus� [63].
Automated endpoint detection system�for LAMP

In an automated end-point detection system, fluorescent SYBR green I�labelled LAMP products are excited by a monochromatic ultraviolet (UV) emitter and a color sensor to detect the emitted fluorescence from the target and displays the results on LCD. This method used for rapid detection as a point-of-care diagnosis. This advanced automated detection system used for detection of salmomella contaminats in foods [46].
Centrifugal LAMP microdevice

The LAMP amplicons are detected by a colorimetric method using Eriochrome Black T (EBT). Centrifugal microdevice is used for dispensing of LAMP reaction solutions automatically and sequentially in to the reaction chambers at controlled RPM. The color changes from purple to sky blue indicate positive reactions. The visible LAMP targeted against Escherichia coli O157: H7,�Salmonella typhimurium�and�Vibrio parahaemolyticus [64].
Applications of the LAMP system
Application of LAMP is a robust and rapid gene amplification technique is vast and employed in several fields including clinical diagnosis of pathogens like bacteria, fungus and viruses. Also used in typing of single nucleotide polymorphism (SNP) typing, inspection of meat adulteration, detection of GMOs, sexing of animal embryos, tumor detection and quantification of template DNA. More and more LAMP based point of care diagnostic devices (POCT) is getting developed for bedside diagnosis. LAMP method was first successfully used in Escherichia coli O157:H7 cells [65] followed by Shigella and enteroinvasive E. coli [66], Mycobacterium sp. [67], Leginonella and the periodontal pathogen Porphyromonas gingivalis [68] and many other pathogenic bacteria. Though the diagnosis of viral diseases is often carried out by histopathology, virus isolation, and various types of ELISAs, nucleic acid hybridization, western blot and PCR, application of LAMP assay in detection of several RNA/DNA viruses of human and animals is on the rise and becoming more popular. LAMP assay was applied first for tomato yellow leaf curl virus detection and found to have 100 times higher sensitivity than PCR [69] and now it is employed for several DNA/RNA viruses and found to be superior in terms of sensitivity, specificity, rapidity and simplicity. The ability of avian myeloblastosis virus reverse transcriptase to withstand high temperatures, allows a single tube reaction of both RT and LAMP procedures as one step RT-LAMP assay which has been reported for different RNA viruses of plants animal and human health importance. Usage of LAMP system due to its speed and simplicity is extended to the diagnosis of fungal diseases including chromoblastomycosis in patients or even environmental detection of these infections and also for retrospective studies in archived clinical samples [70]. LAMP assay has been used for the detection of many important protozoan diseases such as Trypanosoma, Babesia and Cryptosporidium [71]. In addition to animal and human health related issues, the LAMP is applicable to detect or monitor the proportion of genetically modified organisms as foreign DNA sequence in crops or other products [71]. LAMP method has also been successfully applied in the identification of embryo sex in bovine pre-implantation embryos.  The technique was found to identify the gender accurately and can be applied in the field [72]. Rapid detection of tumor specific markers by LAMP has been successfully applied to detect metastasis in specimens from patients with gastric carcinoma [73] and to identify also point mutation in the host cell and predict the effects of the anti-cancer drugs [74]. 
LAMP assays for OIE notifiable animal viral diseases
LAMP technology provides widespread applications in diagnostics especially in the field and resource-limited laboratory settings. The LAMP has been developed for the detection of important OIE notifiable viral diseases in animals [75]. Some of the important OIE notifiable diseases in animals are foot and mouth disease, bovine viral diarrhoea, infectious bovine rhinotracheitis, rift valley fever, peste des petits ruminants, lumpy skin disease, bovine leukaemia, bluetongue, classical swine fever, african swine fever, swine vesicular disease, porcine reproductive and respiratory syndrome, avian influenza, infectious bronchitis, infectious bursal disease, newcastle disease, duck hepatitis and west Nile fever. Despite its huge advantages, the LAMP is underused in the field of veterinary but OIE recognizes the importance of the LAMP in their guidelines [76]. The LAMP technology has not been specifically included in OIE manual of diagnostic tests and its use has been limited due to lack of thermostable LAMP kits. But in human applications, tuberculosis and malaria LAMP kits are available in developing countries [77]. For the implementation of veterinary diagnostics in the field, the LAMP technology has to be accepted and promoted by OIE and other agencies. List of some important OIE notifiable livestock and poultry viral diseases for which LAMP assay has been developed and evaluated targeting different structural/non-structural genes is shown in Table 3. LAMP is a simple, rapid and novel assay used for clinical diagnosis and quantification of Capripox virus DNA. The LAMP assay can replace the disadvantages of PCR and other molecular techniques [78].
LAMP assay for pox viral diseases
	Among the different isothermal assays, Loop-mediated isothermal amplification (LAMP) provides a potential �ASSURED� platform for a diagnostic test to be deployable at field diagnostic settings even for pox viral infections. It has been employed over some pox viruses namely orf virus [113], monkey pox virus [114], CMLV [115] and CaPV [116-117] (Tsai et al., 2009; Iizuka et al., 2009; Venkatesan et al., 2012; Das et al., 2012; Murray et al., 2013) using different gene targets. LAMP assays based on P32 [117] (Murray et al., 2013), Poly (A) polymerase [116] (Das et al., 2012), DNA polymerase [118] (Venkatesan et al., 2015) of CaPVs have been optimized and evaluated for rapid, specific and sensitive detection of members of CaPVs and to differentiate SPPV and GTPV [119] (Zhao et al., 2014). These LAMP assays were specific and sensitive comparable to quantitative PCR assays. Similarly, LAMP assays targeting B2L gene [113, 120] (Tsai et al., 2009; Venkatesan et al., 2016), and DNA polymerase gene [121-122] (Li et al., 2013; Venkatesan et al., 2015) have been successfully done to detect ORFV. 
Other applications of LAMP 
The LAMP assay technique used for species identification and sex determination. The species identification is based on the growth hormone GH 1 gene and the male-specific marker (OtY2m; GU181208) for sex identification [123].The food allergen of soyabean and celergy (Apium graveolens) in the food products could be specifically and rabidly detected by the developed LAMP assay [124,125]. The LAMP assay was developed based on the conserved region of cytochrome b of mitochondrial DNA for the detection of imported ostrich meat with beef meat or low quality meat or with wild ostrich meat [126]. The phage immuno LAMP assay was developed to detect Organophosphorus (OP) pesticides in the food [127]. The LAMP assay also developed based on drug resistance of benzimidazole fungicides is related to the mutation of the �-tubulin gene in Sclerotinia sclerotiorum [128]. The LAMP assay was developed to detect genetically modified crops in the trading field. The cry1Ab gene was transferred into genomic DNA of plants to acquire insect resistance. The high specific and sensitive LAMP assay was developed for detecting the cry1Ab gene in transgenic rice [129].
Stability of LAMP reagents 
In�lyophilized LAMP mixture, it requires only addition of DNA sample and nuclease-free water and kept for incubation in water bath or heat block. Based on the addition of specific primers to a reaction mixture, it is not easy to develop a generalized LAMP kit. A LAMP reaction consists of an enzyme and its buffer, dNTPs, MgSO4, betaine, primers, and the detection dye and it only requires re-constitution and sample addition [130]. LAMP reagents are stable at 25�C and 37�C. Bst DNA polymerase can amplify DNA when it is even not kept at freezing temperatures. So, the application of LAMP in the field is highly possible because it is not affected by ambient temperatures [131]. Lyophilized reagents were stable at 4, 25, and 37 �C and there is no need of cold chains for transportation of lyophilized LAMP reagents and at these temperatures, the reagents were stored up to 720 days, 540 days and 42 days, respectively [132]. By this stability, application of LAMP in the field is possible where the cold chain might not be available. The lyophilized reagent mixture remained stable at room temperature for over one and a half months [133]. At  -20, 4, 25 and 30 �C, the stability of the lyophilized reagents for up to one month [134]. Non-reducing sugar trehalose and dialysis of the enzyme preparations used for stabilization of the lyophilized reaction [133]. Various field applicable LAMP kits are available for human and animal applications (Table 4).
General advantages and limitations of the LAMP system
	The LAMP assay runs at the isothermal condition, which eliminates the need for high cost equipment like thermal cyclers. The reaction uses six sets of primers, all of which need to bind to the target DNA, otherwise, no amplification will occur. The detection of the amplified products can be visual by addition of Hydroxyl napthol blue [25] or SYBR green I dye. The assay can be quantitative by measuring the turbidity of the products using a real-time turbidimeter. The assay is time saving as the results can be obtained in an hour and the sensitivity of LAMP is 10-100 fold more than conventional PCR.  Because of the high level of sensitivity, great caution should be exercised to prevent carry-over or cross-contamination that is the major disadvantages of the LAMP and it leads to the false positive results. Further, LAMP is not applicable for molecular biology purpose and cloning [135] as applicable for PCR. Multiplexing approaches of the LAMP are less successful due to the difficulty in experimental design and its procedures [78] and also the identification of a target by the size of the band on a gel is not possible with LAMP because the product of LAMP results in a ladder like pattern [47]. LAMP need 4 to 6 primers targeting 6 or 8 regions within the target sequence and therefore, it is problematic to design a LAMP primer because the primer design has restrictions.
Advantages of closed-tube LAMP assay as a field diagnostic tool
The LAMP does not require more space and instrumentation.
LAMP reactions can be performed in a water bath at a constant temperature between 60-65�C [25].
 Closed-tube LAMP assay method prevents the risk of cross-contamination.
As a closed tube LAMP assay method, it does not require thermal cycler, laminar flow hoods, gel electrophoresis unit, and documentation system [48].
LAMP amplification products can be easily detected based on turbidity and color change by the naked eye in normal room light [9, 25].
Real-time and quantitative.
High specificity, sensitivity and amplification efficiency.
Conclusion and future potential of LAMP
The LAMP is a rapid and simple gene amplification technique used for the quick detection and identification of microbiological infections. LAMPS is valuable and affordable in terms of the cost involved and ease of visualization that can suit the less equipped field diagnostic centers in developed countries. The LAMP has potential application for diagnosis and surveillance of infectious diseases without the requirement of any skilled personnel and sophisticated equipments. Till date, the LAMP assay has been widely used as a diagnostic tool for the detection of human, livestock, and plant diseases including viral origin. It has been employed also in potential detection of genetic diseases like Down syndrome and Thalassemia in addition to food testing and cancer screening. In summary, all of the features of LAMP prove to be a straightforward molecular diagnostic tool beneficial for on-site field diagnosis as a Point of care testing assay and it has potential characteristics that can fulfill all criteria proposed by the WHO/OIE on ideal diagnostics.
Acknowledgments
The authors thank the Director, Indian Veterinary Research Institute for providing necessary facilities to carry out this study and the staff of Pox Virus laboratory, IVRI, Mukteshwar, for their valuable and timely help in carrying out this work. The financial support provided by DBT, India under North-East Twinning program on DBT-NER on Pox project (BT/385/NE/TBP/2012) is also acknowledged.
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Table 1. Comparison of PCR and LAMP assays
FeaturesPCRLAMPTemperature Different temperature Single temperature Time Minimum 3 hoursLess than 1 hourCost Costly Low cost compared to PCR Thermal cycler Needed No need (water bath is needed)Enzyme Taq DNA polymeraseBst DNA polymeraseDenaturation step compulsorynot compulsoryNo. of primersTwo primers are used to amplify the template DNA Four specially designed primers that recognize a total of six distinct sequences on the DNASensitivity/specificity Not 100%100%DNA purity Requires pure DNA templateNo need of pure DNA templateDetection methods Agarose gel electrophoresis (open tube method)Agarose gel electrophoresis, Visual detection methods (closed-tube method)Detection limit Nanogram level of DNAFemtogram level of DNA Detection method Agarose gel electrophoresis Agarose gel electrophoresis (open tube method)
Visual detection (closed-tube method)



Table 2. Comparison of monitoring methods of LAMP assay
Features Turbidity  AGEDNA intercalating dyeColorimetry methodReal time turbidityLFDBiosensorPrinciple Magnesium pyrophosphate precipitateElectrophorectic separation by ethidium bromide Fluorescent dye (open tube method)Colorimetric indicator (closed-tube method)Magnesium pyrophosphate precipitateImmunorecognition, Chromatography, Hybridization, Electro-active indicatorResponse End pointEnd pointEnd pointReal time Real time End pointEnd pointTime 60 min60 min60 min40 min (HNB)
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No cross contaminarion Assessment of length of ampliconsHigh sensitivitySimple, sensitivity and no cross contamination No cross contamination
Automation High specificity High specificity
Automation  DemeritsRequires skilled personnel Cross contamination Cross contaminationInhibition of amplification Homogenicity Cross contamination Cross contamination Reference  [39] [40] [41] [42] [43] [44] [45]



Table 3: Application of LAMP technology for different OIE notifiable viral diseases of livestock and poultry
OIE notifiable viral diseasesTarget gene (s)Reference Foot-and-mouth disease virus (FMDV)3D RNA polymerase, 2B, VP1 of serotype Asia 1, P1 and polyprotein genes [79-83]
  Bovine viral diarrhea virus (BVDV)52 UTR region [84]Rift Valley fever virus (RVFV)L RNA segment [85]Peste des petits ruminants virus (PPRV)Matrix gene [86]Lumpy skin disease virus (LSDV)poly(A) polymerase small subunit (VP39) [87]Bovine herpesvirus-1 (BHV-1)Glycoprotein E gene [88]Bovine leukemia virus (BLV)LTR region [89]Bluetongue virus (BTV)VP2 gene [90]Classical swine fever virus (CSFV)E2 and NS5B genes[91,92]African swine fever virus (ASFV)topoisomerase II gene [93]Swine vesicular disease virus (SVDV)Conserved  3D polymerase [94]Porcine reproductive and respiratory syndrome virus (PRRSV)M gene
open reading frames 1a [95-97]Avian influenza virusHA gene [98]Infectious bronchitis virus (IBV)Nucleocapsid phosphoprotein gene [99]Infectious bursal disease virus (IBDV)VP3, VP2 and VP5 genes [100,101]Newcastle disease virus (NDV)Fusion protein  [102,103]Duck hepatitis virus (DHV-1)Conserved 3D gene/2C gene [104,105]West Nile virus (WNV)Envelope gene [11]RSV- [106]Porcine circovirus type 3ORF2 [107]Hepatitis B virus- [108]Human Zika VirusNS5 [109]Coxsackievirus B3VP1 gene [110]Herpesvirus of turkey   HVT070 gene [111]Influenza virus H6, H9HA  [112]Table 4. Closed tube field applicable LAMP assays available for different human/animal applications
Purpose                          Kits Human health LAMP TB Kit
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