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Results and Discussion

Senior High School
University of Santo Tomas
In Partial Fulfillment
Of the Requirements for
Practical Research 2
by
JUAN CARLOS DE BOURBON

JUAN CARLOS DE LA BOURBON
JUAN DE LA CRUZ
JUAN DELA PAZ
JUAN MIGUEL CRUZ
JUANITA JUANITO
CARLITO CARLOS
MARCH 22, 2019

ABSTRACT
Early monitoring strategies for diseases is an integral part in shrimp farm

management, particularly for pathogens like the White Spot Syndrome Virus.
However current molecular diagnostic tools such as the conventional one-step PCR
is too expensive and laborious for shrimp farmers to perform. To address the
problem, a loop-mediated isothermal amplification (LAMP)–based kit specific for
WSSV was developed. The kit contains specially designed DNA extraction
procedures and primers suitable for LAMP detection of WSSV. A locally fabricated
heat block with fitted black light was also designed to satisfy LAMP reaction
conditions. The protocol and components of the kit makes it simple, affordable and
time efficient, capable of DNA extraction and amplification without a high level of
expertise. The kit’s DNA extraction procedure is almost two (2) times cheaper than
the existing commercial extraction kit. Comparison of the kit with the conventional
one-step PCR showed that the LAMP-based kit was faster and at the same time
more sensitive than its counterpart having a detection limit of 1.363pg of DNA
whereas PCR having only 13.63pg of DNA. Using the kit, WSSV screening was
performed on samples coming from eight (8) different sites across the Philippines.
The DNA samples were extracted from the shrimp samples and tested for WSSV
using the kit then compared to conventional one-step PCR. The results showed that
the developed kit detected 66.47% of the total collected samples with WSSV while
only 29.41% was detected by PCR demonstrating the sensitivity of kit. Initial field
test results have also shown that the heat block’s fitted black light allowed for
convenient visualization of LAMP products on-site. The future commercialization of
the kit would greatly help in improving farm management practices and reduce the
country’s dependence on expensive and imported diagnostic kits.
Keywords: Shrimp farm management, DNA extraction, WSSV, LAMP

CHAPTER 4
RESULTS AND DISCUSSION
4.1 Results
4.1.1 Evaluation of DNA extraction method effectivity and requirements
To properly compare the applicability of the developed DNA
extraction method of the diagnostic kit with the commercially existing kit ZR
Tissue & Insect DNA MicroPrep™ (Zymo Research, USA), there were a
number of metrices analyzed. Generated DNA from both protocols had
varying yield and purity but with comparable applicability to PCR and LAMP.
Both protocols also had different equipment and time completion
requirements. For the developed DNA extraction protocol, cost per reaction
was determined by estimating theoretical labor costs and actual supplies and
materials cost (See Appendix II). Summarized in Table 3 is the direct
comparison of the metrics of both protocols.
Do may only leave hanging spaces like these if you have a figure. As
shown below. If that is not the case, you must always make sure that you do
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Table 4. Comparison of DNA extraction methods
(See Appendix III for costing breakdown)
4.1.2 Analytical specificity
The generated DNA from both the developed DNA extraction
protocol and the commercial kit were subjected to analytical specificity in
PCR and LAMP and tested against non-target Taura Syndrome Virus positive
cDNA template. Results from the test proved the PCR and the LAMP
primers’ specificity to WSSV. No amplification was seen in the non-target
Taura Syndrome Virus positive cDNA template (Unpublished data, 2013)
(Figure 3). It likewise provided evidence that the generated DNA from the
developed protocol can be subjected to PCR and LAMP same with the
commercial kit.

Figure 3. Analytical specificity. A.) Analytical Specificity of PCR primers viewed in 1% A.G.E. B.)
Analytical Specificity of LAMP primers viewed under 2% A.G.E. C.) Analytical Specificity of LAMP
primers viewed using sybr safe stain. (1) DNA from developed DNA extraction protocol, (2) DNA
from commercial kit, (3) Taura Syndrome Virus template and (N) No template control.
4.1.3 Analytical sensitivity and detection limit
Having been shown that the DNA from the developed protocol
applicable for amplification reactions, it was tested for analytical sensitivity.
The starting DNA product from the developed DNA extraction protocol with
1.363ug/ul concentration was serially diluted ten (10) times and used as
template for the test of analytical sensitivity. For the computation of the
detection limit, the undiluted starting DNA template (10 0 or 1.363ug/ul) was
divided by the dilution factor from the lane where the product can be
visually seen (Highlighted in red boxes – Fig.4A,B,C). For the developed kit,
results showed that LAMP having a detection limit of 1.363pg/ul or 10 -6
dilution (0.0001363ng/ul) of viral DNA was ten (10) times more sensitive
than conventional PCR with only 13.63pg/ul or 10 -5 dilution (0.001363ng/ul)
of viral DNA detection limit (Fig. 4A and 4B). Additionally, LAMP products
were visualized via sybr safe staining until 10-6 dilution of viral DNA (Fig.4C).

Figure 4. Analytical Sensitivity from DNA isolated using developed kit. (A) PCR, (B)
LAMP, (C) LAMP visualized using SYBR-safe stain, (A2) Image J Fluorescence PCR,
(B2) Image J Fluorescence LAMP, (C2) Image J Fluorescence LAMP visualized using
SYBR-safe stain.
Using Image J analysis, fluorescence signal values from PCR gel
bands, LAMP gel product and LAMP products visualized via SYBR safe stain
dye were obtained and plotted against their respective DNA concentrations
(Fig. 4A2, 4B2 and 4C2).

Regression analysis (MS Excel 2013 Data Analysis Toolpak) at 95%
confidence interval (Appendix VI) has shown that there is a linear
relationship between the DNA concentration (ng/ul) and the Fluorescence
signals obtained from the PCR bands. Likewise, fluorescence signals from
LAMP gel product and LAMP products visualized via SYBR safe stain dye
produced similar trends in relation to DNA concentration (ng/ul) (Appendix
VI).
4.1.4 Comparative WSSV Screening using LAMP and conventional PCR
Having established that the DNA from the developed DNA
extraction protocol is applicable for PCR and LAMP with sufficient specificity
to WSSV, it was utilized in the screening of selected shrimp farms in the
Philippines. As shown in Figure 5, Utilizing PCR, WSSV infection was
detected from Bulacan (80.00%), Bataan (64.00%), Cebu (25.00%), Roxas
(20.00%), Davao (20.00%) and Zamboanga (45.00%) while shrimp samples
from Iloilo and General Santos city all tested negative. For LAMP, infection
was detected in all selected sites; Bulacan (93.00%), Bataan (84.00%), Cebu
(62.50%), Iloilo (64.00%), Roxas (72.00%), Davao (50.00%), General Santos
(73.00%) and Zamboanga (45.00%). In summary, from the total one hundred
seventy (170) samples, LAMP was able to detect a total of 66.47% of the

samples positive for WSSV, while conventional PCR was only able to detect
29.41%.
Figure 5. Prevalence of WSSV infection in eight (8) selected Philippine sampling sites detected
using laboratory PCR and LAMP screening. Total number of samples: 170.
McNemar's Test of paired matched data (MS Excel 2013 - PHStat2
Add-in) at 95% Confidence interval reveals that the proportion of samples
diagnosed positive for WSSV is significantly different for PCR and LAMP
(Appendix VII).
In a selected area in Orani, Bataan, DNA was isolated on-field using
the developed DNA extraction procedure. Generated DNA templates were
then amplified via LAMP using the locally fabricated heatblock (UST
Electrical Engineering Department). LAMP products were viewed using a

visualization dye and a fitted portable black light provided with the
heatblock. LAMP was able to diagnose 90% of the samples positive for
WSSV on site (Figure 6). This demonstrates the field applicability of the
diagnostic kit in WSSV screening.
Figure 6. On- field LAMP screening of WSSV viewed using a visual dye and a black light. (P) WSSV
positive control. (N) negative control. Sample 1 – negative for WSSV, Samples 2-10 Positive for
WSSV.
4.2 Discussion
Prior to downstream amplification reactions in diagnostics, it is a prerequisite
of any diagnostic kit to provide DNA with sufficient purity and yield (Abd El-Aal et al.
2010). Leninger’s principles in biochemistry (Leninger et al., 1975) indicate that the
generally accepted absorbance ratio (A260/280) for DNA with ample purity is ≥ 1.8
with a maximum of 2.0. The developed protocol produced a higher concentration of
DNA and optimal purity in comparison with the DNA template produced by the
commercial kit (Table 3). The acquired DNA from the developed protocol had an
absorbance reading of 1.846 (A260/280) indicating that it was indeed sufficiently
pure (Table 3).

Aside from isolation of DNA with excellent yield and purity, one of the most
important goals of DNA extraction according to Boesenberg-Smith et al. (2012) was
the isolation of ample DNA amount suitable for downstream applications such as
nucleic acid amplifications. The developed DNA extraction kit and procedure
provided sufficient quality DNA suitable for PCR and LAMP (Figure 3A and 3B
respectively) at a much faster completion time than the commercially existing kit
(Figure 1). It likewise proved to be simple in terms of equipment requirement and
complexity (Table 3) requiring only a table top centrifuge (≤ 6000rpm) which is
usually readily on stock in reputable suppliers within the Philippines. The commercial
kit on the other hand, required a high speed centrifuge with more than 10,000rpm
as well as an automatic cell lysis machine in the form of Disruptor Genie TM (Zymo
Research, USA) or a standard bench top vortex; these types of machines usually
require time to purchase due to their high complexity with exception of the bench
top vortex (Table 3).
Focusing on practicality and applicability, the developed DNA extraction
protocol proved to be relatively affordable having a cost per reaction almost two (2)
times cheaper than the commercially existing kit (Table 3). In the study of Cuveliers
et al. (2009), he indicated that a commercial kit's cost per reaction is similarly as
important as its produced DNA purity and yield since it is also a limiting factor in the
user’s preference. Raw materials and supplies utilized in the formulation of the

extraction protocol's stock reagents and buffers were readily available in
manufacturers and distributors within the country. The reagent and packaging
preparation of the kit theoretically requires only less than 3 hours as well as minimal
technical skill adding convenience to its reproducibility as reference to prospective
future manufacturers (See Appendix II).
Both the developed DNA extraction protocol together with the commercially
existing kit were based on the concept of solid-phase extraction involving mainly key
four steps such as cell lysis, nucleic acid binding, washing and elution of DNA (Kojima
and Ozawa, 2002). The work of Vandeventer et al. 2012 explained that solid-phase
extraction mainly takes advantage of the binding of DNA and silica surfaces in
different conformations; the use of chaotropic salts (e.g. Guanidium thiocyanate)
plus ethanol can effectively create DNA-accessible silica surface pK a values. These
surfaces display –OH groups where DNA bases can bind forming hydrophobic
bonding. Although the developed protocol was an adaptation from the method of
Boom et al. (1990), it was mainly chosen due to its minimal reagent requirement and
few limitations similar to other solid-phase extraction based protocols. Other
methods of DNA isolation relying mainly on the concepts of organic extraction or
chelex extraction present limitations such as toxic and amplification inhibiting
reagents, time-consuming protocols, and complex machinery requirement (Alonso,
2013) which limit it to laboratory use.

Moreover, the DNA generated from the developed kit encompasses the
values of the commercial kit from a previous study which utilized the same set of
LAMP primers in WSSV detection (Nicolasora et al., 2014). The commercial kit used
by the mentioned study was the WizardTM Genomic DNA Purification kit (Promega,
USA) (Promega Corporation, 2014) which was able to produce detection limit values
of 8.2ng/ul for LAMP and 84.6 ng/ul for PCR, values that were relatively lower than
the values produced by the developed DNA extraction procedure with 1.363pg/ul
and 13.63pg/ul for LAMP and PCR respectively. Furthermore, the mentioned study
provided sufficient DNA to be able to diagnose asymptomatic shrimp WSSV carriers
in selected Philippine sites (Nicolasora et al., 2014) suggesting that the developed
procedure’s higher detection limits would make it sufficiently capable of producing
reliable results.
In addition, it is important to highlight that the results obtained from the
developed diagnostic kit (Figure 4A and 4B) not only agree with the studies
performed within the country (Caipang et al., 2012 and Nicolasora et al., 2014) but
also with the studies from other countries (Kono et al., 2004, Sun et al., 2006,
Nimitphak et al., 2008) which all confirm that LAMP is ten (10) times more sensitive
than conventional PCR.
Even more, the Image J generated fluorescence signals from the analytical
sensitivity tests summarized in Figures 4A2 have shown that in PCR analytical

sensitivity, the decreasing concentration of serially diluted DNA corresponded to
decreasing fluorescence signals. However, for LAMP analytical sensitivity in A.G.E.
(Figure 4B.2) and LAMP visualized using SYBR safe stain (Figure 4C.2), a different
trend was observed. LAMP viewed in A.G.E. had an increasing signal from the
undiluted template (100 or 1363ng/ul DNA) to the first dilution (10 -1 or 13.63ng/ul
DNA) then had peak fluorescence signal at the second dilution (10 -2 or 1.363ng/ul
DNA) and decreased in the succeeding dilutions before reaching its detection limit
(10-6 or 0.0001363ng/ul). A similar trend can be observed for the LAMP products
viewed using SYBR safe stain (Figure 4C.2). The LAMP reaction has been known to
produce large amounts of amplified DNA LAMP products as auto-cycling proceeds
(Notomi et al., 2000). A previous study (Altshuler L. 2006) has shown that the
confinement of too much DNA in the reaction vessel may lead poor DNA synthesis
due to hindered diffusion of large polymerase molecules. Additionally, the ratio of
lesser of target DNA to non-target molecules within the reaction can likewise lead to
inefficient amplification. Unlike PCR which produces same copies of the same gene
region of interest (Alshuler L., 2006), LAMP initially uses the region of interest from
the starting DNA material and produces unique products with varying long DNA
fragments with looping structures (Notomi et al. 2000). The dilution of DNA allowed
for a smaller starting DNA material and a decreased amount of generated LAMP
products in turn giving the BST large fragment DNA polymerase more space for

diffusion. LAMP from A.G.E. visualization have shown that optimal DNA
concentration at the second dilution harboring the highest fluorescence score. On
the other hand, LAMP from SYBR safe stain visualization, optimal DNA concentration
was at the latter third and fourth dilution.
It was also necessary to assess the kit’s performance in shrimp farm screening
to further evaluate its readiness for field setting. The developed kit was again
compared to conventional PCR this time in WSSV screening of farms. WSSV
diagnostic procedures using the PCR technology have been widely practiced in
screening of Philippine farms (Caipang & Aquana, 2010; Maralit et al., 2011;
Magbanua et al., 2000) making it an ideal standard for comparison.
From Figure 5, it can be observed that for all selected sites: Bulacan, Bataan,
Cebu, Roxas, Davao, and Zamboanga, LAMP significantly diagnosed more positive
samples than PCR. Even more, areas that were diagnosed as WSSV negative by
conventional PCR, Iloilo and General Santos city were tested positive in LAMP. In
summary, LAMP was able to diagnose 66.47% of the total collected samples in
comparison to PCR which only diagnosed 29.41% of the samples. This only shows
that the higher analytical sensitivity of LAMP over PCR translates to its sensitivity in
the screening of shrimp farms for WSSV. This notable higher sensitivity can be
explained by the pioneering work of Notomi et al. (2000). It was his work which
indicated that the LAMP technology uses four (4) distinct primers to target six (6)

regions of the desired gene sequence purposely making it more sensitive than
conventional PCR which only uses two (2) primers that target only two (2) end
regions. Additionally, LAMP is able to produce high DNA copy within less than an
hour of amplification at an isothermal temperature in contrast with PCR with takes
two (2) to three (3) hours to complete relying on the three (3) cycling temperatures
(Notomi et al., 2000).
Furthermore, statistical analysis via McNemar’s test at 95% confidence
interval that there is a significant difference between the proportion of samples
diagnosed WSSV positive by PCR and LAMP. The statistical tool is commonly utilized
in the comparison of paired matched data comparing the control and the
experimental test. The test was utilized to effectively compare the standard protocol
PCR (control) and the developed LAMP kit (experimental test) (Lowry R., 2016)
(Appendix VI).
The specialized Bst DNA polymerase in LAMP makes it possible to facilitate
amplification via auto-cycling strand displacement DNA synthesis. Auto-cycling allows
the DNA amplification process to proceed at an isothermal temperature (Notomi et
al., 2000) for a short amount of time. This minimal requirement allows the
technology to be achievable with the use of a simple waterbath or a heatblock which
normally maintains one temperature at a time contrary to PCR which requires an
expensive thermalcycler to proceed (Chen et al., 2010).

Collaborators from the UST Electrical Engineering department took advantage
of LAMP’s minimal requirements by locally fabricating a heat source for the
developed kit’s LAMP assay (See Appendix I). In an initial field test experiment in
Bataan, results have revealed that the developed kit utilizing the heat block was
capable of producing results on field with high specificity diagnosing 90% (9/10) of
the samples with no false-positives via dye visualization (Figure 6). This only
demonstrates the robustness of the developed kit and heat block as it can produce
results in laboratory (Figure 5) and field settings (Figure 6). It’s also important to note
that a portable black light was fitted with the heat block for convenient and safe
visualization of results (Figure 6). This provides an inexpensive alternative to A.G.E.
which often requires an electrophoresis apparatus as well as an UV-light enabled
viewer in results viewing in PCR (Altshuler, 2006) as well as in LAMP (Caipang et al.,
2012; Kono et al., 2004; Maralit et al., 2012 and Nicolasora et al., 2014).
Focusing on LAMP naked eye result visualization, other previous studies
involving WSSV detection of Philippine shrimp samples (Caipang et al., 2012, Maralit
et al., 2012, and Nicolasora et al. 2014) have either utilized white precipitate
formation or use of DNA-intercalating dyes viewed under UV illumination as an
alternative to agarose gel electrophoresis (A.G.E.). According to Mori et al. (2001),
the polymerization of DNA by Bst Polymerase enables the release of pyrophosphate
ions from deoxyribonucleotide triphosphates (dNTPS) as a by-product. These in turn

bind to the magnesium ions from the reaction buffers to form a white precipitate
(Abdul-Ghani et al., 2012; Tanner and Evans, 2014; Mori and Notomi, 2009 and Mori
et al.,2001) that cause the solution to become turbid prior to centrifugation (Le Roux
et al., 2009). Although the method does not require any additional dye or reagent to
complete and can prevent cross-contamination with the elimination of reagent
transfer, the turbidity and white precipitate formation can remain stable only for a
short period of time (Almasi et al., 2013).
On the other hand, the method of fluorescent DNA-intercalating dye usage
shows increased sensitivity values when compared with turbidity measurements
(Soli et al., 2013). However, it often requires the usage of UV light illumination which
can be harmful with direct or indirect exposure (Katoch, 2011). Since the maximum
exposure limit of the human eye is 280nm, UV light’s mid-range (280-315 nm) or far
(200-280 nm) UV wavelength can already cause retinal damage to its user (Katoch,
2011). The study’s utilization of a black light instead of a UV light, makes it ideal for
safe application in routine farm diagnostics since black light’s 400nm wavelength
borders near UV and visible light making it basically safe for the human eye (United
Nuclear Scientific, 2016).
Finally, this is the first study to utilize a Philippine made heat block together
with the developed LAMP-based diagnostic kit for detection of WSSV. The developed
diagnostic kit’s low cost per sample DNA extraction together with its quick and

simple procedure make it a potentially marketable product. The safe result
visualization in the initial field testing showcases the developed kit’s field
applicability in routine shrimp farm diagnostics. Once commercialized, the developed
kit’s low price would compete with existing commercial diagnostic kits lowering the
price of diagnostics in the Philippines. This would in turn aid the regulating and
monitoring agencies in promoting cheaper and more effective routine diagnostics for
the industry thereby preventing uncontrolled disease outbreaks.
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CHAPTER 5

SUMMARY AND CONCLUSION
The developed DNA extraction protocol’s low equipment requirement,
minimal protocol complexity and time completion proved to be advantageous over
the commercially existing kit producing DNA that was usable for downstream
amplification reactions. Likewise, the protocol’s production cost and time provided a
cost per reaction value significantly lower than the commercial kit. In addition, the
developed diagnostic kit as a whole validates previous LAMP studies in achieving
analytical sensitivity values 10 times more than conventional PCR. Furthermore,
WSSV screening results showed that sensitivity values translated into screening
efficiency with LAMP detecting 66.47% and PCR detecting only 29.41% of the
samples. Additionally, preliminary field test result also suggested that the kit can be
successfully applied in field settings with the use of safe and sustainable black light
results visualization.
To conclude, the study was able to develop a LAMP-based diagnostic kit
from cheap and readily available reagents providing a DNA extraction protocol
suitable for PCR and LAMP. The developed kit’s performance was sufficiently
comparable with the commercially kit not only in shrimp farm screening but also in
terms of practicality, time-efficiency and convenience in usage. The developed kit

was also proven to be more sensitive than conventional one-step PCR diagnosing
significantly more samples.

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Results and Discussion

JUAN CARLOS DE BOURBON

MARCH 22, 2019

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Early monitoring strategies for diseases is an integral part in shrimp farm

Keywords: Shrimp farm management, DNA extraction, WSSV, LAMP

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RESULTS AND DISCUSSION

4.1.1 Evaluation of DNA extraction method effectivity and requirements

To properly compare the applicability of the developed DNA

number of metrices analyzed. Generated DNA from both protocols had

Both protocols also had different equipment and time completion

materials cost (See Appendix II). Summarized in Table 3 is the direct

comparison of the metrics of both protocols.

not leave spaces like these

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Table 4. Comparison of DNA extraction methods

(See Appendix III for costing breakdown)

4.1.2 Analytical specificity

The generated DNA from both the developed DNA extraction

protocol and the commercial kit were subjected to analytical specificity in

primers’ specificity to WSSV. No amplification was seen in the non-target

Taura Syndrome Virus positive cDNA template (Unpublished data, 2013)

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4.1.3 Analytical sensitivity and detection limit

applicable for amplification reactions, it was tested for analytical sensitivity.

results showed that LAMP having a detection limit of 1.363pg/ul or 10 -6

than conventional PCR with only 13.63pg/ul or 10 -5 dilution (0.001363ng/ul)

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Using Image J analysis, fluorescence signal values from PCR gel

(Fig. 4A2, 4B2 and 4C2).

Regression analysis (MS Excel 2013 Data Analysis Toolpak) at 95%

confidence interval (Appendix VI) has shown that there is a linear

relationship between the DNA concentration (ng/ul) and the Fluorescence

produced similar trends in relation to DNA concentration (ng/ul) (Appendix

4.1.4 Comparative WSSV Screening using LAMP and conventional PCR

Having established that the DNA from the developed DNA

to WSSV, it was utilized in the screening of selected shrimp farms in the

Philippines. As shown in Figure 5, Utilizing PCR, WSSV infection was

detected from Bulacan (80.00%), Bataan (64.00%), Cebu (25.00%), Roxas

(20.00%), Davao (20.00%) and Zamboanga (45.00%) while shrimp samples

(62.50%), Iloilo (64.00%), Roxas (72.00%), Davao (50.00%), General Santos

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McNemar's Test of paired matched data (MS Excel 2013 - PHStat2

Add-in) at 95% Confidence interval reveals that the proportion of samples

In a selected area in Orani, Bataan, DNA was isolated on-field using

the developed DNA extraction procedure. Generated DNA templates were

Electrical Engineering Department). LAMP products were viewed using a

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diagnostic kit in WSSV screening.

Prior to downstream amplification reactions in diagnostics, it is a prerequisite

with a maximum of 2.0. The developed protocol produced a higher concentration of

absorbance reading of 1.846 (A260/280) indicating that it was indeed sufficiently

pure (Table 3).

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important goals of DNA extraction according to Boesenberg-Smith et al. (2012) was

(Figure 1). It likewise proved to be simple in terms of equipment requirement and

complexity (Table 3) requiring only a table top centrifuge (≤ 6000rpm) which is

top vortex (Table 3).

Focusing on practicality and applicability, the developed DNA extraction