SERAMBI BIOLOGI e-ISSN: 2722-2829

Primer Design and in Silico PCR for Detection Shigella Sp. on

Refilled Water Samples
Deratih Bunga Purwakasih, Afifatul Achyar*
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang, West
Sumatera, Indonesia
*Corresponding author: afifatul.achyar@fmipa.unp.ac.id

Abstract. Refillable drinking water is an effort to meet the needs of drinking water for
consumption by modern society. One of the quality parameters of drinking water suitable for
consumption is not contaminated with Escherichia coli, Salmonella sp. andShigella sp. Conventional
microbiological tests were often conducted for the detection of water pathogenic bacteria,
including culturing the bacteria in medium culture and biochemical assay, but this method
requires a long working time and expensive costs. Currently, Polymerase Chain Reactions (PCR)
can be an alternative method because it has high accuracy. Primers are one of the important
components of PCR so they must be specifically designed to ensure the success of DNA
amplification. This study aimed to obtain the specific sequence of PCR primer for detection
Shigella-contaminated refillable drinking water sample and conducted the in-silico PCR. Primers
were designed in silico using Primer BLAST in NCBI (National Center for Biotechnology
Information) and Geneious Prime for in silico PCR. To get a Shigella-specific primer, pairwise
alignment of Shigella sp. (NC_004337.2) and E. coli (NC_000913.3) was performed and specific
sequence of Shigella was used as primer candidates. The result of this study was the Shigella-
specific sequence PCR primer forward 5'-GCTAATGAAAATGGCGCTGT-3' and reverse 5'-
AGCCGACGGTTTGAAGTTAC-3' with PCR product length of 815 bp.

Key words: Refillable drinking water, Shigella sp., Primer Design, in silico PCR.
This is an open access article distributed under the Creative Commons 4.0 Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. ©2020 by author.

Introduction
Refillable drinking water is an effort to fulfill the need for drinking water for consumption by
modern society. Refillable drinking water is usually obtained from refill drinking water depots which is an
alternative because the price is much cheaper than branded bottled drinking water products. This is what
causes drinking water depots in various cities in Indonesia to grow very rapidly (Bambang et al., 2014).
Drinking water with quality that does not meet standards will have a negative impact on health due to the
presence of pathogenic bacteria which make drinking water a medium for spreading. Based on research
conducted by Nuria, Rosyid & Sumantri (2009) based on the Decree of the Minister of Health
No.907/Menkes/SK/VII/2002 states that one of the parameters of the quality of drinking water suitable for
consumption is not contaminated with Escherichia coli bacteria, Salmonella sp. and Shigella sp.
Shigella sp. including Gram-negative bacteria in the form of rods, not spores, not encapsulated, and not
motile, measuring 0.5-3 µm, growing optimally at 37oC and pH 7.4 (Parija, 2012; Sari, 2012). Shigella sp. can be
spread through faecal-oral directly or through contaminated food and drink (Atmiati, 2012). Shigella multiplies
without invasion in the jejunum and then produces a toxin called Shiga toxin. The toxin will bind to the receptor
and cause the activation of the process of water secretion resulting in watery diarrhea (Venkatesan et al., 2012),
this condition is called Shigellosis, were sufferers experience diarrhea. Clinical symptoms of shigellosis range
from mild diarrhea to severe dysentery, depending on the serotype of Shigella sp. leading to infection and host
immunity. The survey conducted by Herwana et al. (2010) in 612 children aged 0-12 years who suffered from
diarrhea, the result was 9.3% caused by Shigella sp. and 63.2% (36/57) of them were S. flexneri.
Microbiological tests are often carried out for the detection of waterborne pathogenic bacteria
conventionally, namely by means of culture and biochemical properties. However, this method requires a
long processing time and is expensive, because further tests must be carried out, namely biochemical,
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serological and Vero toxicity tests. In addition, the results obtained are also non-specific. Therefore, it is
necessary to develop methods for detecting these aquatic pathogens. Currently, the Polymerase Chain
Reactions (PCR) method can be an alternative because it has high accuracy (Radji et al., 2010).
The PCR method is a revolutionary method developed by Kary Mullis in the 1980s. The working
principle of PCR is based on the ability of the DNA polymerase enzyme to synthesize new DNA strands which
are complementary to template DNA (Mullis et al., 1994). The main components of the PCR reaction consist of:
template DNA or template DNA; designed primer; DNA polymerase enzymes; and nucleotides (dNTP or
deoxynucleoside triphosphate) (Handoyo & Ari, 2001).
Primers are short pieces of single-stranded DNA that are complementary to the target gene sequence. Primers
must be specifically designed to ensure the success of the DNA amplification process in the PCR method
(Diss, 2003). Design to obtain a primer that meets the criteria for amplification can be done in silico, namely
designing or designing primers with the help of a computer program (software). One of the commonly used
software for literature studies and Primary BLASTING is a BLAST feature in NCBI (National Center for
Biotechnology Information). BLAST works to analyze the specificity of the primers that have been designed,
using GenBank DNA database data (Munsinin et al., 2018).
This study aims to obtain specific PCR primer sequences for examining refill drinking water samples contaminated
with Shigella and to simulate them with in silico PCR. The PCR primers are expected to be used for molecular
detection of the presence of Shigella sp. on refill drinking water samples. This needs to be done for the accuracy
of the results and to save processing time allocation.

Materials and Methods

Material
The material used in this study was the complete genome sequence of Shigella sp. (NC_004337.2)
and E. coli (NC_000913.3) downloaded from GenBank NCBI (http://www.ncbi.nlm.nih.gov). The tool used is
Geneious Prime based bioinformatic software for designing primers and PCR in silico.
Method
The gene sequence of Shigella sp. and Escherichia coli are so similar that it would be difficult to find specific genes
for each species. Therefore, the strategy for designing Shigella-specific primers was carried out by pairwise
alignment of the complete genome sequence of Shigella sp. and E. coli using the NCBI BLAST. Species-specific
regions are used as primary candidates.
The designed primer must meet the criteria of an ideal primer, namely having a nucleotide length ranging from 18-
30 bp, having a base composition of 40-60% G and C, Tm between forward and reverse primers not having a
difference of more than 50C, and not forming a secondary structure (hairpin or self-dimers). The primary candidates
that have been obtained from the results of pairwise alignment are checked using Geneious Prime software to ensure
that they meet the ideal primary criteria above.
The specificity of the designed primers was checked using the Primer BLAST tools on the NCBI website. The
purpose of this check is to ensure that the primer that has been designed will only amplify the specific target gene
of Shigella sp. The primer pairs obtained were simulated by in silico PCR using Geneious Prime.

Results and Discussion

In this study, an in-silico primer design was carried out using BLAST primers on NCBI. Sequence alignment is a
process of juxtaposing two or more sequences so that the differences and similarities will be obvious (Xiong, 2006).
Shigella sp. sequences and E. coli have a similarity of 99%, meaning that the nucleotide arrangement is very similar.
To obtain Shigella-specific primers, Pairwise Alignment of Shigella sp. (NC_004337.2) and E. coli (NC_000913.3).
Figures 1 and 2 show fragments of the results of the complete genome alignment of the two bacteria in specific
regions. This pairwise alignment produces a forward primer 5'-GCTAATGAAAATGGCGCTGT-3' and a reverse
primer 5'-AGCCGAGGTTTGAAGTTAC -3'. These two primers are the best primers among the primary
candidates.

Figure 1. Forward Primary Candidates Result of Pairwise Alignment Shigella sp.NC_004337.2 (Query) and E. coli NC_000913.3
(Sbjct)

Figure 2. Primary Candidate Reverse Pairwise Alignment Results Shigella sp.NC_004337.2 (Query) and E. coli NC_000913.3
(Sbjct)
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In silico PCR is performed to simulate and predict PCR results and minimize errors when PCR is performed in vitro.
PCR was carried out in silico using Geneious Primer software. Geneious Prime is a user-friendly bioinformatics
application that integrates genomic data analysis. This software can also be used to ensure that the primary
candidates meet the ideal criteria for amplification (Kearse et al., 2012).

Table 1. Primary Characteristics of Design Results

No. Characteristics Primer DNA Fold Amplicon Size
1.

815 bp
2.

In general, the ideal primer length is between 18 and 30 oligonucleotides. If the length of the primer is less than 18
bases, it will easily cause mis priming so that the primer will stick to unwanted places. Primer lengths of more than
30 bases will show low specificity and result in a hybridization process with other primers that will inhibit the
formation of DNA polymerization (Maitri et al., 2016). Table 1 shows the length of the forward primer and reverse
primer each measuring 20 bp, which means that they already have an ideal primary length so that they can reduce
the risk of misplacing.

Tm (melting temperature) is the temperature when the double strands of DNA will separate and the recommended
Tm value is between 50-65°C. The difference Tm between the forward and reverse primers is best not more than
50. If the difference is more than 50 it will cause a decrease in the efficiency of the amplification process (Sasmito
et al., 2014). According to the primary criteria in Table 1, the difference between the forward and reverse primary
Tm is not more than 50, which is only 1.10, so it is good for the amplification process.

The percentage of GC is the content of bases G (guanine) and C (cytosine) which can affect the Tm of a primer and
the bonds between strands in DNA. The recommended GC content ranges from 40% to 60%. The high GC content
makes it difficult to separate the double-stranded strands in the primer and template DNA. A primer that has a low
GC content will not be able to stick effectively to the target so there will be a decrease in the efficiency of the PCR
process (Handoyo & Ari, 2001).

PCR reactions should not contain secondary structures such as hairpins and dimers. Secondary structures result in
primers not being able to attach to the DNA template. Hairpins are structures built by polynucleic acid co-bases
between complementary sequences of a single strand of either DNA or RNA. Hairpins should be avoided in
designing primers, but sometimes it is very difficult to obtain primers that do not form hairpin or loop structures.
Self-dimer occurs when bonds are formed in two similar primers (forward primer with forward primer or reverse
primer with reverse primer). Self-dimer should be low or even non-existent for truly ideal primary results (Handoyo
& Ari, 2001). Table 1 shows that the primer has been designed according to the requirements to meet the ideal
criteria.

Primer specificity was rechecked with Primer BLAST-tool at NCBI. This is so that primers that meet the ideal
criteria will indeed only amplify Shigella sp. Table 2 shows how the specificity of the primers has been designed.
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Table 2. Primer Specificity Test using Primer BLAST
Target template Primer Shigella F group Shigella R
Shigella flexneri Yes
Shigella boydii Yes
Shigella sonnei Yes*1
Shigella dysenteriae Yes*1
Escherichia coli Yes*1
Salmonella typhi Not
Salmonella bongori Not
Salmonella enterica Not
Legionella pneumophila Not
Vibrio cholerae Not
* Minimum number of mismatches with the target primary sequence

From the table it can be seen that the primer will only amplify Shigella flexneri and Shigella boydii. For
Shigella sonnei, Shigella dysenteriae and Escherichia coli also have almost the same sequence, but there is a
mismatch at the 3' end of one of the primers so it still cannot be amplified. For a common primer size of 20 bp the
mismatch at the 3' end will affect the annealing temperature so that the template does not stick to the target gene
(Simsek & Adnan, 2000)..
In silico PCR was performed for the primer pair with the genome sequence of Shigella sp. (NC_004337.2)
using Geneious Prime software (Figure 3). In silico PCR results showed that the primer pair could attach to the
genome sequence of Shigella sp. (NC_004337.2) at 3,295,600 – 3,295,619 nucleotides for Shigella F primers and
at 3,296,395 - 3,296,414 nucleotides for Shigella R primers. This primer pair will amplify the yhbV and yhbW gene
regions located on Chromosomal DNA, so that an 815 bp amplicon will be obtained. These results are consistent
with predictions of PCR product size with Primer BLAST. The use of in silico PCR is to predict and simulate the
attachment of primer sequences to template DNA sequences, so as to minimize errors when performing PCR in
vitro.

Figure 3. In silico PCR with Shigella F and Shigella R primer pairs using Geneious Prime

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Conclusion
The primers that were successfully designed met the requirements as ideal primers for the PCR amplification
process. The primer design made in silico produced a forward primer of 5'-GCTAATGAAAATGGCGCTGT-
3' and a reverse primer of 5'-AGCCGACGGTTTGAAGTTAC -3'. The primer pairs succeeded in amplifying
the yhbV and yhbW gene regions with a size of 815 bp according to the in-silico analysis results produced on
Geneious Prime and BLAST results on NCBI.

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