4. 4/14/2021
4
Quality laboratory testing is crucial
Need of the hour is to promote rational, cost-effective
diagnostic methods
Control and eradication of
disease….
..starts with diagnosis
Each year in sub-Saharan Africa, ∼12 million people
die……
Animals too…..
Infectious diseases-HIV infection, malaria, and
tuberculosis….. endemic, epidemic and pandemic
HS, FMD, PPR, CSF, BT, CD, Parvo, Trypanosomiasis….
Other…TB, JD, Brucellosis…
5. 4/14/2021
5
Ideal Diagnostic test
WHO recommends that an IDEAL DIAGNOSTIC TEST suitable
for developing, underdeveloped and undeveloped countries
should be
“ASSURED”
☻ Affordable
☻ Sensitive
☻ Specific
☻ User-friendly
☻ Robust and rapid
☻ Equipment free
☻Deliverable to the end user
6. Easy-to-Use
Affordable
Unspecific clinical
signs
Low specificity
Low sensitivity
4/14/2021
6
Common Diagnostic Methods
Laboratory
Methods
Field Methods Clinical signs/symptoms
Smear
CMT
RBPT
TB/JD skin test
In-vitro culture
Ag and Ab detection
ELISA
Agglutination tests
Precipitation tests
Molecular tests
PCR
DNA Hybridization
High specificity
High sensitivity
Complicated
Expensive
Time consuming
7. 4/14/2021
7
Cont..
Limitation of available diagnostics
Either less sensitive, cumbersome or require
highly sophisticated instruments
User friendly and cost effective diagnostics with higher
sensitivity and specificity are the NEED OF THE
PRESENT HOUR
Isothermal amplification methods
promising field for development of pen-side
diagnostics
8. 4/14/2021
8
Isothermal amplification Assays
• Includes NASBA, HDA, MDA, RPA, RCA, LAMP, CPA and PSR
• Most require multiple enzymes (three or more) and rigorous optimization
• But.....
RCA, LAMP , CPA and PSR can be efficiently performed at a constant
temperature using one enzyme
• However....
RCA method can only amplify circular DNA
• LAMP require initial denaturation for the satisfactory results
• PSR is novel and unique
11. 4/14/2021
11
What it require..??
Primers (PSR-FP and PRS-RP)
Bst polymerase enzyme
Bacillus stearothermophilus
strand displacement
activity
Optimum temperature (55-
65ºC)
greater tolerance to
inhibitors (Kaneko et al. 2007;
Francois et al. 2011; Soleimani et
al.2013)
dNTPs
Instrument
Betaine (N,N,N-trimethylglycine)
Reduce base stacking
destabilize the DNA helix
facilitate strand separation
MgSO4
forms magnesium
pyrophosphate
Buffer
12. 4/14/2021
12
Primer Design
Forward Primer (F) 5'- acgattcgtacatagaagtatag-GTTTGATCGTCAGGGATGG-
Backward Primer (B) 5'- gatatgaagatacatgcttagca-GCATAAGTCGCAATCCCCG
F
B
N
Nr
As simple as PCR primers de
22. 4/14/2021
22
Sensitivity of PSR
Lane 1: 5.6x10-1 ng
Lane 2: 5.6x10-2 ng
Lane 3: 5.6x10-3 ng
Lane 4:5.6x10-4 ng
Lane 5: 5.6x10-5 ng
Lane 6: 5.6x10-6 ng
Lane 7: 5.6x10-7 ng
1
2
3
4
5
6
Conventional PCR PSR assay
Real time-PCR
Visual detection after addition of SYBR-I dye
24. 4/14/2021
24
Properties PCR PSR LAMP
Denaturation Required Not required Only initial step
Primers 2 2 6
Annealing-
Extension
3 steps at different
temp
Isothermal (60-65C) Isothermal (60-
65C)
Time required 2-3 hours 30-60 min 30-60 min
Optimization Easy Easy Difficult
Post
amplification
Needs AGE No need No need
Sensitivity ng of DNA fg of DNA fg of DNA
Instrument Needs
sophisticated and
costly instrument
No need of
expensive
instruments
No need of
expensive
instruments
Template
preparation
Requires
/Impurities hinder
the amplification
No or minimum
processing
No or minimum
processing
Move on PCR, Here’s come PSR
26. 4/14/2021
26
Detection limit of PSR was 6.9 pg/ml within 1 h, 10-fold higher than that of PCR
(69.0 pg/ml)
Fourteen non-C. albicans yeast strains were negative for detection, which
indicated the specificity of PSR assay was 100%
effective C. albicans detection assay was developed that has a great potential
for clinical screening and point-of-care testing.
28. 4/14/2021
28
The detection limit of parvoviral DNA by PSR, real-time PCR, and LAMP was 5
9 10-6 ng, while PCR was able to detect up to as 5 9 10-5 ng of parvoviral DNA.
Thus, a tenfold increase in sensitivity was observed with the newly developed
PSR when compared with conventional PCR
29. Department of Veterinary Microbiology, DUVASU, Mathura 4/14/2021
29
two temperature step approach was employed: an initial denaturation step of
95 °C for 5 min
The developed PSR technique is 100 fold more sensitive than conventional
PCR and comparable to real-time PCR.
31. 4/14/2021
31
Future Perspectives
Addition of internal set of primer can further increase the
sensitivity
Introduction of RE sites on primer Ft and Bt will aid in
sequencing of the product and other molecular biology
experiments
Technique could also be applied in biosensor-based
analytical instruments
Can be coupled with LFA for multiplex diagnosis
Has a great potential to be adopted in pen-side test
Most of the developing and undeveloped countries are in Africa and Asia. Another common characterstic of the two regions is the prevalence of diseases. A lot of time, money and energy have been spent fighting diaseases rather than building the economy. The state organs will also use their resources to deal with disease outbreaks, common disease includes polio, malaria, yellow fever and HIV
Lack of effective point of care diagnostic tests applicable in resource-poor endemic areas is a critical barrier to effective treatment and control of infectious diseases” (Njiru , 2012)
“Diagnosis is important not only for the prescribing of effective drugs for appropriate patients in adequate doses but also for preventing the evolution of resistant microorganisms” (Mori and Notomi, 2009)
The tests should be sensitive
2. Test should be specific
3. Reasonable cost so that the farming community may be benefited
4. Simple protocols to perform
5. Rapidly performed
6. Adopted to any sort of climatic variation
7. Instruments used should be low cost and easy available everywhere
Solely developed by……Academy of Military Medical Sciences, Beijing, China
First reported by Liu et al in 2015 of …….
PSR is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases and amplification of genes
The whole procedure is very simple and rapid which in the amplification can be completed in less than 1 hour under isothermal conditions by incubating the mixture of samples, primers, Bst polymerase with strand displacement activity and substrates all the in a single tube
PSR provides high amplification efficiency, with DNA being amplified 109–1010 copies in 30–60 min at 65℃, isothermally
No need for a step to denature double stranded into a single stranded form
Amplification and detection of gene can be completed in a single step
Because the efficacy of PCR amplification may be affected by the presence of a number of inhibitors of Taq polymerase enzyme in the serum such as EDTA, phenol, polyamines, polysaccharides and calcium alginate, DNA extraction is required before amplification
It has been shown that PSR exhibits less sensitivity to inhibitory substances present in biological samples than PCR. This robustness of PSR against inhibitors can contribute to saving the time and cost required for sample processing steps
advantage of amplifying the target DNA from partially processed and/or non-processed sample .This exclusion of DNA extraction step not only shortens the reaction time and result interpretation but also eliminates the chance of contamination .
The amplification efficiency is extremely high
Reduced total cost- not require special reagents or sophisticated equipments
In this study, we describe a novel isothermal nucleic acid amplification method, named Polymerase
Spiral Reaction (PSR). Only one pair of primers and one enzyme are needed to launch the PSR reaction,
just like PCR. Besides, since the reaction is performed at a constant temperature, an energy intensive
thermal cycler is not needed. Moreover, the positive results could be determined through a visual color
change. The PSR method is appropriate for on-site and point of care testing.
Bst enzyme
considered as the “brain box” of the technique
Bacillus stearothermophilus
contains the 5´→3´ polymerase activity
lacks 5´→3´ exonuclease activity
exhibit greater tolerance to inhibitors typically found in diagnostic specimen (e,g blood, humic acid), which is an advantage as compared to PCR polymerase
optimal temperature for Bst polymerase is between 55-65°C
At temperatures above 70°C, enzyme activity is significantly decreased while at 80°C the enzyme is inactivated
Cost Rs 4200 for 8000 units/ml
Magnesium sulphate
Magnesium sulphate which forms magnesium pyrophosphate during the course of the reaction which enables to visualize the result based on the turbidity formed.
•Betaine (N,N,N-trimethylglycine)
Betaine is used to reduce base stacking and to increase not only the overall rate of reaction but also target selectivity by significantly reducing amplification of irrelevant sequences.
It also destabilize the DNA helix and negates the need for heat denaturation of the template.
It is theorized that reaction temperatures of 65°C in conjunction with betaine are sufficient to facilitate strand separation; however, the exact mechanism is unknown
Increased target selectivity with a significant reduction of irrelevent sequences
The forward and reverse Tab primer sequences are reverse to each other at their 5’ end (Nr and N), whereas their 3’
end sequences are complementary to their respective target nucleic acid sequences.
The uppercase 3’ sequences of the forward
primer (F) and reverse primer (B) are complementary to the target blaNDM-1 gene sequence (position
32922–32941, 33115–33097, GenBank accession: 11027496). The lowercase 5’ sequence of the forward
primer (Nr) is reverse to the lowercase 5’ sequence of the reverse primer (N). Nr and N sequences were
abstracted from a botanic gene.
When the temperature reaches 61–65 °C, the double-stranded structure of template DNA unlocks due to the presence of Betaine.
F segment of the Ft primer anneals to one single-stranded DNA and extends
the B segment of the Bt primer hybridizes to it (structure 3) and extends (structure 4).
The double strands of structure 4 melts and forms a single chain (structure 5).
Sequence Nr and N are reverse to each other, and sequence Nr and Nrc are complementary to each other.
Thus, sequence N and Nrc are reverse and complementary to each other.
Structure 5 would curl to structure 6.
The 3’ end of Nrc would continue to extend and finally forms a spiral structure (structure 7).
Similarly, the extension mechanism of another single-stranded chain in the right diagram is the same as the left diagram.
With the time and temperature preset, gene
amplification will occur, and the detection can be
done by simultaneously monitoring the white
turbidity caused by the existence of magnesium
pyrophosphate, the amplification by-products
In the DNA amplification process by DNA polymerase, pyrophosphate ions are produced as a
by-product from the reaction substrate deoxyribonucleotide triphosphates (dNTPs). The calcein
in the reaction mixture initially combines with manganous ion (Mn2+) so as to remain quenched.
When the amplification reaction proceeds, manganous ion is deprived of calcein by the
generated pyrophosphate ion (P2O7
4- ), which results in the emission of fluorescence. And the
free calcein is apt to combine with magnesium ion (Mg2+) in the reaction mixture, so that it
strengthens the fluorescence emission.
combines the advantage of PCR in which only one pair of
primers is needed and isothermal amplification techniques such as RCR and 3SR.