MICROBIAL DIAGNOSTICS: REPORT 1
N1006934
INTRODUCTION
The last two decades have seen a growing trend towards the techniques of bacterial
pathogen genomics and genome mining, receiving much attention due to its pivotal role
in public health (Armstrong, 2019). Research convincingly shows that complex
epidemics are being transformed by next-generation sequencing and analytics,
featuring novel clues regarding pathogen development and transmission (Scheffler,
2013). For example, NRP secondary metabolite recognition from microorganisms like S.
aureus has been made possible due to genome mining, providing new biochemical
epiphany about NRP biochemistry (Nadine Ziemert, 2016). Polymerase chain reaction
(PCR) based assays allows the detection of bacterial strain at the species level by
targeting specific genes and designing primers to amplify the target regions efficiently
(Francis Martineau, 2001). A thorough overview of molecular & PCR diagnostic assays,
and rapid methods to detect methicillin-resistant Staphylococcus aureus (MRSA) which
have been commercially available as of date, has been given (Angela S. Rossney, 2008).
The present report establishes a set of criteria for genome mining to identify pathogen-
specific (i.e., Staphylococcus aureus) target genes for PCR diagnostic assays. On the basis
of various bioinformatic tools, it then describes the selection of a unique gene of the
strain S. aureus FRP3757 for further use.
METHODOLOGY
Step-wise analysis of various bioinformatic tools used in this report are described as
follows.
International sequence data bases such as National centre for biotechnology
Information (NCBI), GenBank, European Molecular Biology Laboratory (EMBL),
Ribosomal Database Project (RDP), SILVA, DNA Data Bank of Japan (DDBJ) and
Greengenes are used to submit and retrieve genomic sequences for identification of
genes and whole genomes (Pevsner, 2015). They can be accessed in a variety of
sequence file formats such as FASTQ, FASTA, GenBank and EMBL. To download a gene
sequence from the NCBI website, click on” all databases” and select nucleotides; in the
search box either type in the name of your organism of interest or its accession number.
For example, “Staphylococcus aureus[orgn] USA300_FPR3757 not plasmid” or
“CP000255.1”
, Enter organism
and strain name
Click & Select
in the search
Nucleotide in
box.
All Databases
Figure 1
Download the genome sequences of S. aureus FPR3757 strain in FASTA/GenBank
format.
Click on “Send to” &
“Complete Record “and
choose destination as
“File”. Download in
FASTA format.
Figure 2
Repeat the same steps for obtaining the genome sequence of S. aureus MRSA252
(methicillin resistant SA) strain (Accession Number:NC_002952.2).
Now to obtain the unique genes for both the strains, the Comprehensive Antibiotic
Resistance Database (CARD) and Virulence finder 2.0 (Centre for Genomic
Epidemiology) are used.
, In CARD, go to Analyse and use the resistance gene identifier (RGI) to upload the FASTA
sequence of the strain FPR3757 (as CARD only recognizes FASTA format files).
Click on
“Analyze”.
Figure 3
Click on
“RGI”.
Figure 4
N1006934
INTRODUCTION
The last two decades have seen a growing trend towards the techniques of bacterial
pathogen genomics and genome mining, receiving much attention due to its pivotal role
in public health (Armstrong, 2019). Research convincingly shows that complex
epidemics are being transformed by next-generation sequencing and analytics,
featuring novel clues regarding pathogen development and transmission (Scheffler,
2013). For example, NRP secondary metabolite recognition from microorganisms like S.
aureus has been made possible due to genome mining, providing new biochemical
epiphany about NRP biochemistry (Nadine Ziemert, 2016). Polymerase chain reaction
(PCR) based assays allows the detection of bacterial strain at the species level by
targeting specific genes and designing primers to amplify the target regions efficiently
(Francis Martineau, 2001). A thorough overview of molecular & PCR diagnostic assays,
and rapid methods to detect methicillin-resistant Staphylococcus aureus (MRSA) which
have been commercially available as of date, has been given (Angela S. Rossney, 2008).
The present report establishes a set of criteria for genome mining to identify pathogen-
specific (i.e., Staphylococcus aureus) target genes for PCR diagnostic assays. On the basis
of various bioinformatic tools, it then describes the selection of a unique gene of the
strain S. aureus FRP3757 for further use.
METHODOLOGY
Step-wise analysis of various bioinformatic tools used in this report are described as
follows.
International sequence data bases such as National centre for biotechnology
Information (NCBI), GenBank, European Molecular Biology Laboratory (EMBL),
Ribosomal Database Project (RDP), SILVA, DNA Data Bank of Japan (DDBJ) and
Greengenes are used to submit and retrieve genomic sequences for identification of
genes and whole genomes (Pevsner, 2015). They can be accessed in a variety of
sequence file formats such as FASTQ, FASTA, GenBank and EMBL. To download a gene
sequence from the NCBI website, click on” all databases” and select nucleotides; in the
search box either type in the name of your organism of interest or its accession number.
For example, “Staphylococcus aureus[orgn] USA300_FPR3757 not plasmid” or
“CP000255.1”
, Enter organism
and strain name
Click & Select
in the search
Nucleotide in
box.
All Databases
Figure 1
Download the genome sequences of S. aureus FPR3757 strain in FASTA/GenBank
format.
Click on “Send to” &
“Complete Record “and
choose destination as
“File”. Download in
FASTA format.
Figure 2
Repeat the same steps for obtaining the genome sequence of S. aureus MRSA252
(methicillin resistant SA) strain (Accession Number:NC_002952.2).
Now to obtain the unique genes for both the strains, the Comprehensive Antibiotic
Resistance Database (CARD) and Virulence finder 2.0 (Centre for Genomic
Epidemiology) are used.
, In CARD, go to Analyse and use the resistance gene identifier (RGI) to upload the FASTA
sequence of the strain FPR3757 (as CARD only recognizes FASTA format files).
Click on
“Analyze”.
Figure 3
Click on
“RGI”.
Figure 4
