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Optimization of Mcfarland Turbidity Standards Value in Determining Template

DNA as Reference in Salmonella Typhimurium Atcc 14028 Test Using Real-Time
Pcr (QPCR)

Article  in  PalArch's Journal of Archaeology of Egypt/ Egyptology · October 2020

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS

VALUE IN DETERMINING TEMPLATE DNA AS REFERENCE
IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING
REAL-TIME PCR (QPCR)

Zulfiayu Sapiun , Vyani Kamba and Sukmawati A. Damiti

Department of Pharmacy, Health Polytechnic of Gorontalo, Gorontalo City
96123, Indonesia
Alfi Sophian and Abinawanto
Department of Biology, Faculty of Mathematics and Natural Sciences,
Universitas Indonesia. Depok 16424, Indonesia
Muindar and Alfi Sophian
Laboratorium of Microbiology, National Agency of Drug and Food Control in
Gorontalo, Gorontalo City 96123, Indonesia.
Herman Luawo
Department of Nursing, Health Polytechnic of Gorontalo, Gorontalo City
96123, Indonesia

Zulfiayu Sapiun, Alfi Sophian, Abinawanto, Muindar, Vyani Kamba, Sukmawati A.

Damiti, and Herman Luawo : Optimization of Mcfarland Turbidity Standards Value in
Determining Template DNA as Reference in Salmonella Typhimurium Atcc 14028 Test
Using Real-Time Pcr (QPCR) -- Palarch’s Journal of Archaeology of Egypt/Egyptology.
ISSN 17 (6), 1567-214x

Keywords: McFarland, Standard, Real-Time PCR, DNA, Template.

ABSTRACT
Background and objectives: An optimization of McFarland standards in determining the DNA template
in Salmonella typhimurium ATCC 14028 test using real-time PCR (qPCR) was conducted to specify
which McFarland standard value that is appropriate to be used as reference of template DNA in the
particular test.
Materials and Methods: Conducted in the Microbiology Laboratory of Center for Food and Drug
Control (CFDC) in Gorontalo, this research relied on qualitative analysis using real-time PCR with SYBR
Green method. Moreover, DNA isolation technique involved direct method by extracting one ose needle
from Nutrient Agar (NA) slant medium. Further, the data analysis was conducted in two steps: 1)
amplification of DNA template and observation of cycle threshold (Ct) and melting temperature (Tm)
values; 2) statistical analysis by one-way ANOVA.

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

Results: The results indicate that the average Ct value is in 15.18 - 15.53. Statistical analysis on Ct value
indicates Fcount < Ftable, (or 0.109 < F critical 1.541) with α = 0.05; signifying that McFarland turbidity
standard value does not influence the Ct value. Further, as the result of Tm analysis indicates, the average
value of Tm is in 84.8 - 85.7. Statistical analysis on Tm value indicates Fcount < Ftable, (or 0.281 < F
critical 1.541) with α = 0.05; signifying that McFarland turbidity standard value does not influence the
Tm value.
Conclusion: All in all, this research concludes that the reference template DNA for qPCR analysis by
McFarland turbidity standard is 0.5 - 2.

INTRODUCTION
PCR has been used to detect DNA for several organisms (1–3). The International
Standard Organization (ISO) has published the standard as reference for PCR test to
detect pathogen bacteria (4). Studies on Salmonella, however, only begun to involve
real-time PCR test since 2002 (5–8). Piknova et al (9) conducted study on 75 types of
Salmonella spp by real-time PCR with SYBR green method; the study result reports
that 100% were detected. Another study on Salmonella suggests the similar result; the
study was carried out on 51 Salmonella isolations (10). There are at least 12 widely
used genes (himA, invA, stn, hns, spvC, oriC, ompC, fimC, agfA, sefC, misL, phoP,
and phoQ) to detect Salmonella by PCR test (11–14).
On the other hand, studies on molecular analysis towards pathogen bacteria employing
boiling and direct methods as the isolation method have shown no discussion on purity
and concentration analysis; leading to no standardization of template DNA. In attempt
of solving the problem, this study intends to identify and determine the reference of
template DNA for DNA molecular analysis of bacteria using real-time PCR based on
McFarland turbidity standard unit as the reference of colony amount of the template
DNA.
McFarland standard was employed as the analysis pattern on turbidity of
microorganism suspensions in determining the amount of colony. The standard was
made from the combination of Barium chloride and sulfuric acid that creates
flocculation (15). Applied in microbiological test, the McFarland standard aims to
predict the amount of bacterial colony culture dissolved in a series of dilution.
Salmonella indicates that there is no exact amount determined on colony cultures
added as the template DNA(16). On that ground, this research aims to determine the
colony standard as template DNA in molecular analysis towards Salmonella
typhimurium ATCC 14028 with McFarland turbidity standard as the reference for the
template DNA colony.
MATERIALS AND METHODS
Research Materials
The research materials involved phase-1 Salmonella typhimurium ATCC 14028 as the
bacterial base, nutrient agar slant, four different concentrates of McFarland standards
(0.5, 1, 1.5, and 2), physiological NaCl solution, QuantiNova SYBR green qPCR kit
(Qiagen), forward primer, and reverse primer.
The forward primer comprised (5’-ATC AGT ACC AGT CGT CTT ATC TTG AT-
3’), while the reverse primer comprised (5’-TCT GTT TAC CGG GCA TAC CAT-3’)
(17).

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

Sample Preparation
The bacterial base (Salmonella typhimurium ATCC 14028) were spread into NA slant
and incubated in 35-37oC for 24 hours. Moreover, four tubes were prepared, each
containing 5ml of physiological NaCl solution. An ose needle of bacterial base was
gradually applied and homogenized by vortex into the NaCl tubes. Each tube was
standardized by different McFarland standard (0.5, 1, 1.5, and 2). The McFarland-
standardized tubes containing colonies were treated as template DNA.
Master Mix Setup
The total volume of master mix is 10 μL, consisting of 5 μL master mix SYBR green,
1 μL forward primer, 1 μL primer reverse, 1 μL water-free Rnase and 2 µL DNA
template.
Cycling and Melt Curve Analysis
Cycling and melt curve analysis was carried out using qPCR (QIAGEN 5 Plex) with
the 2 step cycling method: Denaturation 95oC for 45 seconds and Annealing /
Extention 60oC for 45 seconds.
Data Analysis
The data were analyzed by qPCR employing SYBR Green method; it was to identify
two criteria, i.e., Ct analysis (identifying and comparing the Ct value of amplification
in a given cycle during which a target gene is identified) and Tm analysis (identifying
and comparing any melt pattern at occurs at certain temperatures).
Statistical Analysis
One way ANOVA was conducted as the statistical analysis technique to determine
whether or not the value of McFarland turbidity standard implicates toward Ct and Tm
values based on qPCR amplification result.
RESULTS

Figure 1. Ct analysis result: Black = negative control; Red = 0.5 McFarland; Green =
1 McFarland; Yellow = 1.5 McFarland; Blue = 2 McFarland.
Table 1. Average Ct value from qPCR amplification result
No. McFarland Standard Average Ct value
1 0.5 15.42
2 1.0 15.18
3 1.5 15.53
4 2.0 15.21

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

Table 2. Average Tm value from qPCR amplification result

No. McFarland Standard Average Tm value
1 0.5 85.7
2 1.0 85.5
3 1.5 84.8
4 2.0 85.5

Figure 2. Tm analysis result: Black = negative control; Red = 0.5 McFarland; Green =
1 McFarland; Yellow = 1.5 McFarland; Blue = 2 McFarland.
DISCUSSION
qPCR Analysis
Employing QuantiNova SYBR green qPCR kit, the analysis aims to identify cDNA
and gDNA with highest specificity. The QuantiNova DNA polymerase were stored in
inactive position in lower temperature. The chemical compounds involves mechanism
that simplifies the working system without overlooking the sensitivity and efficiency
of PCR. The inert dye in QuantiNova yellow dilution buffer works as the visual
indicator informing that every reaction was correctly arranged. The bluish color from
the master mixture increases the visibility of contents in reaction tube; the color
changes to green after the application of template DNA diluted in QuantiNova yellow
buffer (18).
Delibato et al indicate that SYBR green method is treated as the preliminary method
for screening in detecting Salmonella in molecular manner (19). As suggested by the
result of qPCR analysis, every sample analyzed was amplificated well; given that the
Ct and Tm were amplificated but the negative control was not. The qPCR method
involves 2-step cycling: Denaturation 95oC for 45 seconds and Annealing / Extention
60oC for 45 seconds.
The invA forward primer comprised (5’-ATC AGT ACC AGT CGT CTT ATC TTG
AT-3’), while the reverse primer comprised (5’-TCT GTT TAC CGG GCA TAC
CAT-3’) [10]; the primer was developed and validated by Malorny et al. (10) to detect
Salmonella. Validation is an essential stage in developing a standard method in order
to provide accurate results as reference (18). Study on 75 types of Salmonella spp by
real-time PCR with SYBR green method; the study result reports that 100% were
detected (9).

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

Cycling Analysis (Ct Value)

Ct value is the final amount of amplicon produced. In quantitative PCR, the DNA
amplification is monitored in each PCR cycle. When the DNA is in log linear
amplification phase, the amount of fluorescence increases above the background. The
point in which fluorescence is quantifiable is called Cycle Threshold (Ct). Using
several dilutions from the given standard DNA, a standard curve is generated from the
log concentration towards Ct (19).
The analysis result indicates that average Ct value of samples with different
McFarland standard is as follows: 0.5 McFarland samples are detected in Ct of 15.42;
1 McFarland samples are detected in Ct of 15.18; 1.5 McFarland samples are detected
in Ct of 15.53; and 2 McFarland samples are detected in Ct of 15.21. The average Ct
value is displayed in the following table 1.
Ct value is the point in which fluorescence signals meet their threshold point.
Analysis of Ct value is aimed to identify the amount of preliminary DNA copies, since
the Ct value is inversely proportional with the amount of targets (20).
Melt Curve (Tm Value) Analysis
The analysis is conducted by identifying any changes in fluorescence during labelling
process of double-stranded DNA (dsDNA) by dye. Once the dsDNA is labelled and
heated, a sudden decrease of fluorescence occurs; this condition is referred to as
temperature melt (Tm) (Real-time PCR Handbook). Theoretically, the Tm of a PCR
product is achieved when 50% of the product was separated. The Tm point resulted is
highly specified for each PCR product and is dependent on the product length and its
nucleotide type .
The analysis result indicates that average Tm value of samples with different
McFarland standard is as follows: 0.5 McFarland samples are detected in Tm of 85.7;
1 McFarland samples are detected in Tm of 85.5; 1.5 McFarland samples are detected
in Tm of 84.8; and 2 McFarland samples are detected in Tm of 85.5. The average Tm
value is illustrated in table 2.
The study’s result is different from Delibato et al. (19) in which the detected
Salmonella by melt curve analysis indicates that the Tm value is at 81.29 ±0.20°C.
The different result may be caused by different use of template, as well as different
concentration and primers. According to Life Technology (21), Tm value is dependent
to the primer length, its GC alkali contents, and other factors e.g., different PCR
products. De Medici et al. (22) researched three strains of Salmonella: S.
Typhimurium, S. Infantis and S. London; it is found that the melt analysis does not
provide significant difference between the strain variations.
Tm analysis can only be conducted by employing real-time PCR; it detects any
fluorophore bounded to amplicon. The amplification by SYBR green dye generates
curve that shows the significant increase in Tm value in which it is bound and
amplificated with template DNA. When the melt curve is formed, any decrease in
fluorescence value is visible in form of single strain of DNA (20).
Statistical Analysis The result of statistical analysis by one-way ANOVA shows that:
1) in Ct analysis, it is generated the Fcount < Ftable, (or 0.109 < F critical 1.541) with α =
0.05, signifying that McFarland turbidity standard value does not influence the Ct
value; 2) in Tm analysis, it is indicated that Fcount < Ftable, (or 0.281 < F critical 1.541)
with α = 0.05; signifying that McFarland turbidity standard value does not influence
the Tm value.

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OPTIMIZATION OF MCFARLAND TURBIDITY STANDARDS VALUE IN DETERMINING TEMPLATE DNA AS PJAEE, 17 (6) 2020
REFERENCE IN SALMONELLA TYPHIMURIUM ATCC 14028 TEST USING REAL-TIME PCR (QPCR)

In conclusions, the reference template DNA for qPCR analysis by McFarland turbidity
standard is 0.5 - 2.
ACKNOWLEDGMENTS
The authors would like to thank the staff of the microbiology laboratory and head of
the National Agency of Drug and Food Control (NADFC) in Gorontalo for laboratory
support.
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