LOOP MEDIATED ISOTHERMAL AMPLIFICATION(LAMP)

DR.D. JYOTHI
POSTGRADUATE
MICROBIOLOGY DEPARTMENT
Table of contents

 Introduction
 What is LAMP
 Mechanism of LAMP
 Principle
 Types of LAMP
 Applications of LAMP
 Advantages & dis advantages
 Infectious disease is one of the most concerning health issue world wide.

 To provide patients with effective medical treatment and emergence of

drug resistant strains ,quick & reliable diagnostic techniques are in high
demand.

 Diagnostics using Molecular techniques have emerged as a promising

methodology because of their high sensitivity.
Molecular techniques

 Nucleic acid amplification methods have been very effectively

utilized in Diagnosis of variety of infectious diseases.

 Nucleic acid amplification: Increase in the number of millions of

identical copies of a DNA/RNA target sequence.
NUCLEIC ACID BASED METHODS
NA based
methods

Sequencing and
Hybridization Amplification enzymatic
digestion of NA

PCR Non PCR

Based based

Coupled target
Isothermal
PCR & signal
amplification
amplification

Helicase
LAMP NASBA TMA dependent
amplification
 PCR is the Gold standard method of nucleic acid amplification based molecular
diagnostic applications.

 Modifications------Fluorescent probes for real time amplification detection

Reverse-transcriptase PCR, multiplex PCR, Nested
PCR, etc.

 It still presents certain challenges in resource-poor and non-laboratory settings.

 Drawbacks-- requirement of expensive and sophisticated thermocycler equipment,

trained technical personnel and extended reaction times limits its potential for
being a rapid point of-care diagnostic technique.
LAMP
 A novel and rapid diagnostic method - Loop-mediated isothermal amplification
(LAMP)

 Reliable and robust method for detection and identification of viral and microbial
pathogens.

 LAMP is used for pathogen and/or antimicrobial resistance detection in veterinary

medicine or medical settings.

 LAMP can detect the nucleic acid of parasites, bacteria, fungi, viruses, etc.

 LAMP can be a valuable tool to aid in the detection and management of disease
outbreaks.
 PCR
PCR
VS LAMP LAMP

Reaction occurs at Different temperatures. Requires a Isothermal-at a constant temperature.60 to 65 °C

thermocycler for temperature cycling.

More time, lengthy reaction. Less time-1hr .

Requires two primers. Require 6 primers.

Trained expertise are needed. Little or no technical expertise to run the assay/interpret
results.

Sophisticated laboratory settings. Mobile friendly/No need of sophisticated laboratory

settings.

Not amenable to visual detection. Amenable to visual detection based on turbidity.

More sensitive than PCR.
Non specific annealing of primers. Specific amplification of target DNA even though
presence of non target DNA.
 INTRODUCTION
 The basic method of LAMP was first developed by Notomi et al. (2000).

 Loop-mediated isothermal amplification (LAMP)

 Reaction occurs at one temperature( 65 °C)
 Amplification is rapid.
 Uses DNA polymerase with high strand displacement activity.
 Uses Reverse transcriptase for RNA target sequence or utilizes enzyme
which having both RT & DNA polymerase activity.

 The most commonly used detection signals are either colorimetric or

fluorescent, but they can also be combined with lateral flow or mobile phone
detection systems.
LAMP WORK FLOW
 WORKING MECHANISM OF LAMP
 LAMP is performed at an isothermal constant temperature, most commonly at
65 °C.
 LAMP primers (two or three primer pairs)
 Forward inner primer FIP
 Reverse inner primers BIP
 Forward and reverse external primers (F3 and B3)
 Third primer pair - Forward loop primers (LF)
Reverse loop primer(BF)
 Detection

AMPLIFIED
TARGET DNA

pH CHANGE
USING pH
TURBIDIMETRIC FLUORESCENCE COLORIMETRIC SENSITIVE
DYES PHENOL
RED

dsDNA-SPECIFIC CALCEIN DYE

MAGNESIUM DYES SUCH AS
PYROPHOSPHATE SYBR GREEN, YELLOW TO
PICOGREEN GREEN

Farhan Jalees Ahmad et .al https://doi.org/10.1016/j.crmicr.2022.100120

 Types of LAMP assays

LAMP

LAMP-on-a-chip Digital/droplet
Single target (microfluidic- LAMP (single-
Multiplex LAMP
LAMP based and paper- cell/molecule
based chips) LAMP)

MERT-LAMP
LEC-LAMP (loop
(multiple Melting
primer
endonuclease temperature-
endonuclease
restriction real- dependent LAMP
cleavage)
time LAMP assay)
1. Single target LAMP

 LAMP (DNA target)

• Regular LAMP assay that has been used to detect a wide
range of pathogens using Bst DNA polymerase, a strand
displacing DNA polymerase enzyme.

 RT-LAMP (RNA target)

• Single-step LAMP platform using reverse transcriptase
enzyme to transcribe RNA targets into cDNA before Bst
DNA polymerase starts target amplification.

• Clinical diagnosis and surveillance of several disease

outbreaks in developing countries.
 RT LAMP -Recent LAMP assay development for human viruses.
PATHOGEN TIME SENSITIVITY DETECTION
METHOD
Zika 20 min 10− 5 PFU Colorimetric

Ebola 15–20 min 256 copies Real-time fluorometer

HIV 60 min 104 RNA copies/mL RT Quencher probe
Influenza 60 min 0.1 RNA copies Phenol-red pH indicator

Hepatitis B 15 min 2.2fg/µl Fluorometer

Dengue 15 min 0.8 fg/µl Colorimetric (SYBR
Green I)
HPV 60 min 10 copies Colorimetric (SYBR
Green I)
H1N1 <40 10 copies turbidimeter, UV
min RNA
Real-time
West Nile virus 17 min 0.1 PFU Real-time turbidimeter

Farhan Jalees Ahmad et .al https://doi.org/10.1016/j.crmicr.2022.100120

2. Multiplex LAMP
 Multiplex LAMP (mLAMP) utilizes more than one gene to detect
multiple target sequences simultaneously, which enhances the
specificity and accuracy of this technique.
 It has also been applied to differentiate between divergent strains or
serotypes of the same pathogen.
 Major merits of the method are
 Short analysis time (<30 min),
 Quantifying a large dynamic range, ability to use unprocessed
samples (saliva, tissue)
 High sensitivity (1–10 copies),
 Compatibility with advanced technological devices (smartphones).
 Recently Kim et al. (2021) reported mLAMP assay to differentiate between
tuberculosis (MTB) and non-tuberculosis Mycobacterium (NTM) species by
designing primers for IS6110 genes that are specific for MTB and rpoB genes
that are common to both.

 A study by Mahony et al. (2013) detection of Influenza A/H1, A/H3 and B

demonstrated. targeted the matrix genes for Influenza H1 and H3 and NS1 gene
for Influenza B .

 Nurul Najian et al. (2016) fabricated a mLAMP based biosensor to detect

pathogenic Leptospira using gold nanoparticle-based detection in a lateral flow
dipstick.
 Recent Studies using Multiplex-LAMP
Pathogen Targets References Sensitivity Specificity References

Colistin resistant mcr-1 to mcr-5 104 copies/µL for 100% (Zhong et al., 2019)
bacteria mcr-1, mcr-2, mcr-
4 and mcr-5,
and105 copies/µL
for mcr-3
SARS-CoV-2 100% Open reading frame genes 105 copies 100% (Kim et al., 2019)
(Kim et al., 2019) 1b (ORF1b) and N
(nucleocapsid)
Foot-and mouth O, A, Asia1, SAT1, 98.0% 98.1% (Yamazaki et al.,
disease virus SAT2, SAT3 2013)
Influenza A (A/ H1 segment 7 of 94.62% for 100% (Jang et al., 2020)
and A/ H3) and influenza A and the influenza A and
influenza B nucleoprotein gene 97.50% for
of influenza B influenza B

Farhan Jalees Ahmad et .al https://doi.org/10.1016/j.crmicr.2022.100120

LEC-LAMP (loop primer endonuclease cleavage)

 Modified LAMP to detect multiple targets in one assay reaction.

 The 5′-end of each forward loop primer for each target sequence is
modified with an a basic site flanked by different fluorescent dyes
and quenchers.
 The LEC-LAMP assay is also modified to include endonuclease IV
enzyme to help differentiate single nucleotide polymorphisms .
 MERT-LAMP (multiple endonuclease restriction real-time LAMP assay)
 Similar endonuclease-based LAMP where the inner forward primers
contain the endonuclease recognition site and are labelled with a fluorophore at
the 5′ end with a suitable quencher in the middle of the sequence.

 Melting temperature-dependent LAMP –

 Simultaneous detection of Salmonella and Vibrio parahaemolyticus.
 The LAMP assay contained the two primers sets to specifically amplify
both bacterial species with a subsequent melting curve analysis to
differentiate the detected target sequence.
 3. LAMP-on-a-chip (microfluidic-based and paper-based chips)

 Mobile point-of-care innovative LAMP to be used for bedside detection

combining sample preparation methods with molecular detection.

 A point-of-care LAMP device was also developed to allow lysis, mixing and
LAMP amplification on a microfluidic chip.

 The system is composed of the chip and a small analyser that is supported with a
portable battery and is controlled by a smartphone, allowing the real-time
fluorescent signal to be transmitted and displayed on the smartphone.

 Farhan Jalees Ahmad et .al https://doi.org/10.1016/j.crmicr.2022.100120

4. Digital/droplet LAMP (single-cell/molecule LAMP)

Recently developed platform ,mirroring digital PCR and employs single-cell generation
techniques with LAMP detection.
Droplet LAMP offers detection at a single-molecule level with great potential for
multiplex detection [13]

Farhan Jalees Ahmad et .al https://doi.org/10.1016/j.crmicr.2022.100120

 ADVANTAGES

 Sensitive & Specific.

 Low cost.

 User-friendly results.

 Mobile friendly.

 Can be used with different sample types.

 Can be used in remote or outbreak settings.

 Real-time when coupled with fluorometric detection.

 LIMITATIONS

 Differentiation of active infection versus asymptomatic

colonization can be difficult.

 Does not differentiate between live/dead target cells.

 Primer–primer interactions could affect specificity.

 Multiplexing methods are less developed.

 Quantification is relative and performance criteria can be

difficult to establish.
 RECENT DEVELOPMENT

 LAMP has been widely adopted as a diagnostic tool to detect various

pathogens, including Salmonella, Escherichia coli, Campylobacter,
Mycobacterium, influenza, HIV, etc.

 Detect parasites such as Babesia gibsoni in dogs and African

trypanosomes.

 To differentiate dengue virus serotypes.

 Detect Streptococcus pneumoniae virulence factor-encoding gene

autolysin (lytA).
 Omni Amp polymerase- has strand displacement and reverse transcriptase
activity, making it suitable for both DNA and RNA targets.

 Omni Amp polymerase allowed amplification at higher temperatures (66–72

°C), which resulted in 20% faster detection in comparison to Bst polymerase.

 LAMP - Detect severe acute respiratory syndrome coronavirus 2 (SARS-

CoV-2) utilizing a portable device that allows wireless communication of
detection data to smartphones.
 FUTURE DEVELOPMENTS

 Facilitating rapid multiplex detection of multiple pathogens in resource-limited

settings or in outbreaks will be an essential tool for the future of diagnostics.

 Mobile isothermal amplification devices simultaneous with user-friendly universal

smartphone detection algorithms in outbreaks areas will also be key to the success
of future molecular detection systems.

 Development of point-of-care detection and microfluidics designs to facilitate a

lab-on-a-chip approach, and to allow this molecular tool to be more effectively and
rapidly used in outbreak settings.

 These developments will be essential for designing cost-effective, rapid and mobile
LAMP assays that facilitate the simultaneous detection of multiple pathogens,
particularly in resource-limited settings.
 APPLICATIONS OF LAMP

 Detect a wide range of pathogens from simple Escherichia coli (Teh

et al., 2014) to the newest SARS-CoV-2 (Chaouch, 2021)

 Neglected tropical diseases (NTD), a group of around 20 diseases

caused by bacteria, virus, fungi or parasites and mostly prevalent in
the poorest regions of the world (WHO 2021).

 Due to poor resources and limited lab settings in these areas, LAMP
has proven to be a revolutionary technique for therapy monitoring,
early disease diagnosis and contact tracing.
 To differentiate closely related species and avoid mis diagnosis.
EX: Mycobacterium leprae, M. tuberculosis (Selvarajah et al., 2020) (specifically
amplify the 16S rRNA target sequence of M. leprae ) (Garg et al., 2021)

 LAMP-based assays -can detect low levels of Plasmodium parasite in malaria.

 Neglected parasitic diseases have been shown to be detected with high

sensitivity and specificity through conventional LAMP.
 TB-LAMP - In the form of a diagnostic kit, has been recommended by WHO
(WHO 2021)

 Rapid LAMP assays --- Respiratory diseases like pneumonia caused by

Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae,
Staphylococcus aureus and Stenotrophomonas maltophilia. (Vergara et al., 2020)
THANK YOU