THP 411-Kul 13

Aplikasi Teknik Biomolekuler untuk

pengembangan metode deteksi cepat (rapid
methods) dan kit deteksi: Lateral Flow
Assays (LFIAs) dan Loop-mediated
Isothermal Amplification (LAMP)

Dr.rer.nat. Asadatun Abdullah

D E PA RT E M E N T E K N O L O G I H A S I L P E R A I R A N
FA K U LTA S P E R I K A N A N D A N I L M U K E L A U TA N
I N S T I T U T P E RTA N I A N B O G O R , 2 0 2 0
We will see this Video first
https://www.youtube.com/watch?v=z07CK-4JoFo
 Lateral Flow Assay (LFA)
• “Lateral flow assay is rapid, easy to use, specific and robust
screening test methods.
• This method employs basic principles from the subjects of
biology, chemistry, physics and engineering fields.
• “…rely on lateral fluid flow through a membrane..”
• Lateral flow (immuno)assay is a technology that is currently
widely applied”

Lata et al. 2013; Posthuma-Trumpie et al. 2009

Dr.rer.nat. Asadatun Abdullah/Kuliah 13-2020

 LFA Basic Principle
 “The basic principle underlying in LFA is:

1. Pergerakan dari liquid sample

2. containing the target analyte along a strip of polymeric
material
3. by the action of capillary flow across various reactive zones
where molecules have been attached that exert more or
less specific interactions with the analytes….”
Lata et al. 2013; Posthuma-Trumpie et al. 2009; Anfossi et al. 2013

Dr.rer.nat. Asadatun Abdullah/Kuliah 13-2020

 Reveal for Histamine is a single-

step lateral low assay based on a

competitive immunoassay format

intended for the visual screening of

histamine in scrombroid species of

fish, such as tuna and mahi-mahi.

www.neogen.com

Dr.rer.nat. Asadatun Abdullah/Kuliah 13-2020

 R e v e a l 2 .0 R e v e a l 2 .0
R e v e a l 2 .0F o r P S P Fo r PS P

Fo r P S P  “Reveal 2.0 for PSP (paralytic shellfish

poisoning) is a 5 minute lateral flow

assay.

 PSP causing toxins can be produced

by dinoflagellates (Alexandrium and

Gymnodinium)
In t e n d e d U s e In t e n d e d U s e
R e v e a l 2 . 0 f o r P S P ( p a r a l y t i c s h e l l fi s h p o i s o n i n g ) i s a 5 m i n u t e l aRteev er aal 2l Once

.0fl foo rwP S aP s( psaar ayly t ic s h e llfi s h p o is o n in g ) is a 5 m i n u t e la t e r a l fl o w a s s a y
the toxin is present in the
t h a t d e t e c t s s a x i t o x i n e q u i v a l e n t s . P a r a l y t i c S h e l l fi s h P o i s o n i n gt h ca tadue t se ci tns gs a xti ot o xx i in ne qs u iv a le n t s . P a r a ly t ic S h e llfi s h P o is o n in g c a u s i n g t o x i n s
c a n b e p r o d u c e d b y d i n o fl a g e l l a t e s o f d i f f e r e n t g e n e r a i n c l u d i n g A l e x a n d r iy ud m
c a n b e p r o d u c e d b in o fl a g e lla t e s o f d iffe r e n t g e n e r a i n c lu d i n g A le x a n d r iu m
a n d G y m n o d i n i u m . O n c e t h e t o x i n i s p r e s e n t i n t h e s h e l l fi s h t i s s u eshellfish
a n d G y m n i t c a n n on tissue
o d i n iu m . O ct e t h e t o x initis cannot
p r e s e n t i n t hbe e s h e destroyed
llfi s h t is s u e it c a n n o t
b e
b e d e s t r o y e d b y h e a t d u r i n g c o o k i n g . R e v e a l 2 .0 fo r P S P i s i n t e n d e d fo r t h e d e s t r o y e d b y h e a t d u r in g c o o k in g . R e v e a l 2 .0 fo r P S P is in t e n d e d fo r t h e
q u a lit a
q u a l i t a t i v e s c r e e n i n g o f s h e l l fi s h P S T ’s , w h i c h p r o d u c e a p o s i t i v e r e s u l t w i t ht iv e s c r e e n in g o f s h e llfi s h P S T ’s , w h ic h p r o d u c e a p o s it iv e r e s u lt w it h
by heat during cooking.
s a m p l e s c o n t a i n i n g a p p r o x i m a t e l y 8 0 0 p p b S T X e q u i v a l e n t s o rs a gm rp ele as ct oenrt .a i n i n g a p p r o x im a t e ly 8 0 0 p p b S T X e q u iv a le n t s o r g r e a t e r .

P ro d u c t S p e c ifi c a t io n s
S e n s it iv it y : 800 p p b ST X 
P ro d u c t S p e c ifi c a t io n s
Reveal 2.0 for PSP
S e n s it iv it y :
is intended for the
80 0 p p b ST X
T e s t in g t im e : 5 m in u te s T e st in g t im e : 5 m in u te s
Sto ra ge: R o o m t e m p e r a tu r e ( 1 8 – 3 0 °C S) t o r a g e :qualitative screening.”
R o o m t e m p e r a tu r e ( 1 8 – 3 0 °C )
T e s t s p e r k it : 24 T e s t s p e r k it : 24

R e s u lts R e s u lt s
In te n d e d U s e N e g a t iv e
N e g a t iv e www.neogen.com
R e v e a l 2 .0 fo r P S P ( p a r a ly t ic s h e llfi s h p o is o n in g ) is a 5 m in u t e la t e r a l fl o w a s s a y
t h a t d e t e c t s s a x it o x in e q u iv a le n t s . P a r a ly t ic S h e llfi s h P o is o n in g c a u s in g t o x in s
c a n b e p r o d u c e d b y d in o fl a g e lla t e s o f d iffe r e n t g e Pn oe sr ita ivine c lu d in g A le x a n d r iu m P o s it iv e

a n d G y m n o d in iu m . O n c e t h e t o x in is p r e s e n t in t h e s h e llfi s h t is s u e it c a n n o t
b e d e s t rAsadatun
Dr.rer.nat. o y e d b y hAbdullah/Kuliah
e a t d u r in g c o o13-2020
k in g . R e v e a l 2 .0 fo r P S P is in t e n d e d fo r t h e
 Commercial Kits for Fish and Seafood Quality Assurance

Biotoxin detection

Test kit price: $27.50

Canadian dollars/single use
test

Source: http://www.jellett.ca

Dr.rer.nat. Asadatun Abdullah/Kuliah 13-2020

 Figure 3

LATERAL FLOW COMPONENTS

Figure 3: Schematic of a generic lateral flow test. The sample pad absorbs the sample and transports the sample to the
conjugate pad. The conjugate pad contains the dried down antibody-nanoparticle conjugate. The nitrocellulose
membrane has test and control lines that show the assay results. The wick pad continues to pull the sample through the
strip at an even rate. All components are assembled on a backing card.
Innova Biosciences Ltd
Babraham Research Campus
We will this video first
https://www.youtube.com/watch?v=o4iJgz9ugy4
Loop-mediated Isothermal Amplification
Video of LAMP
https://www.youtube.com/watch?v=L5zi2P4lggw&t=12s

https://www.youtube.com/watch?v=2EV1sd2V1Js
 Th e N e e d

Ideal diagnostic test suitable for developing should be:

“ Re lia b le ”
☻Affordable
☻ Sensitive
☻ Specific
☻ User-friendly
☻ Robust and rapid
☻ Equipment free
☻ Deliverable to the end user

Eu rop e an Co n fe re n ce on Xy le lla fas t id ios a 2 0 1 7 : fin din g an s w e rs t o a g lo b a l p ro b le m

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 LAMP ( LOOP MEDI ATED I S OTHERMAL AMP LI FI CATI ON )

Originally reported by N o t o m i e t a l in 2000 of EIKEN Chemical

Co. Ltd., Japan

( h t t p :/ / w w w .e ik e n .c o .jp / e n / )

As of 17th May 2017, PubMed database has listed more than 1668
articles on this topic

Eu rope an Con fe re n ce o n Xy le lla fas t id ios a 2 0 1 7 : fin d in g an s w e rs t o a g lob al p rob le m

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 LAMP c h a ra c t e ris t ic s

Bs t DN A p o ly m e ra s e with strand displacement activity at

65℃

No need for a step to denature double stranded into a

single stranded form

The amplification efficiency is extremely high

Reduced total cost not require special reagents or

sophisticated equipment

Eu rop e an Con fe re n ce on Xy le lla fas t id ios a 2 0 1 7 : fin d in g an s w e rs t o a g lob al p roble m

5
LAMP Primers
A Six Primer Mix Produces Optimal LAMP Results

Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html

FIP (Forward Inner Primer) BIP (Backward Inner Primer)

F3 (Forward Outer Primer) B3 (Backward Outer Primer)
FL(Forward Loop Primer) BL(Backward Loop Primer)
LAMP vs. PCR
LAMP Assays are Faster, Simpler Once Developed

PCR LAMP
1. Requires temperature cycling Isothermal – single temperature

2. Requires 2 primers Requires 6 primers

3. Slow: Typically >1hr Rapid: Typically <30 min

4. Typical yield ~ 0.2 g Typical yield ~ 10–20 g

Amenable to visual detection based on
5. Not amenable to visual detection
turbidity etc.
6. Sensitive to sample matrix inhibitors Tolerant to sample matrix inhibitors

7. Can be multiplexed Difficult to multiplex

 LAMP Amplification Detection Methods
Multiple Choices Are Available Depending on Needs

Mg Precipitation Colorimetric Agarose Gel Real Time Turbidity

Factors for Optimization
Multiple Parameters Should be Assessed

• Primers (design, concentration and ratio)

• Enzyme (concentration)

• Reaction temperature

• Mg ion concentration

• Reaction pH

• Additives such as betaine

Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html
References

1. Anfossi L, Baggiani C, Giovannoli C, D’Arco G, Giraudi G (2013) Lateral-flow immunoassays

for mycotoxins and phycotoxins: a review Anal Bioanal Chem 405: 467-480
2. Bonwick, Graham A., and Christopher J. Smith. "Immunoassays: their history, development
and current place in food science and technology." International journal of food science &
technology 39.8 (2004): 817-827.
3. Lata, Kiran, et al. "Lateral flow assay-concept and its applications in food analysis." Indian
Food Industry 32.5 (2013): 22-32.
4. Posthuma-Trumpie GA, Korf J, van Amerongen A (2009) Lateral flow (immuno) assay: its
strengths, weaknesses, opportunities, and threats. A literature survey. Anal Bioanal Chem
393: 569-582
5. Volkov A, Mauk M, Corstjens P, Niedbala RS (2009) Rapid Prototyping of lateral flow assays.
In: Rasooly A, Gerold KE (Eds.), Methods in molecular biology: Biosensors and biodetection
Humana Press, Clifton, pp. 217-235
6. https://www.innovabiosciences.com/resources/applications/lateral-flow-immunoassay/
THANK YOU!!!