Introduction or fluorescent probe are combined with PCR

Introduction

Over the past years, clinical Microbiology has become reliant on the techniques of molecular diagnostic. Frequently in the diagnosis of infectious diseases, the amplification
of nucleic acids are undergone using molecular tests 1.

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Traditionally,
assays based on phenotype rather than the genotype
of pathogens were used but are now
being replaced by current technologies in diagnostic microbiology. Some of the
traditional assays in microbiology diagnostic are
serology, antigenaemia, microscopy, and microbial culture 2. However, despite the
usefulness of these assays new technologies have allowed us to better
understand and analyse the dynamics and
composition of microbial communities. For
example, In past decades, human intestinal bacteria were quantified using quantitative Polymerase Chain Reaction (PCR) amplification of specific sequences 3.

Polymerase Chain
Reaction (PCR) is a technique that was invented in 1984 by Kary Mullis, that amplifies
a region DNA or a gene. This technique later revolutionized
science and paved the way for RT-PCR or real-time
PCR which was later invented 1996 4which allowed the product of PCR to be detected
in real time5.

The visualization
of PCR product occurs in real time when
DNA detection dyes such as syber green or
fluorescent probe are combined with PCR 6. The of PCR steps can be divided into three
processes: denaturing, annealing and extending. During denaturing,
double-stranded DNA is heated above 90 C and separated into two single strands. Then the temperature is then lowered
between 50-60 C to allow oligonucleotide
primers to anneal to DNA template. In the
third step, the temperature is raised between 70-78 C allowing optimal primer
extension; thus, creating a new strand of DNA 6.

Throughout the years conventional
PCR methods have been superseded by RT PCR due to its detection of different
pathogen strains, rapid diagnosis, detection and
quantification of PCR products 1. Also, the close setting of
PCR  reduces the chances of contamination
and provides sensitivity and specificity
in research6.  These various advantages of RT PCR  have
allowed the technique to become well-established and increased drastically. However,
despite several improvements in the increase of accuracy,
there is a limitation to the use of RT-PCR7, 8.

One of the prominent problems that
lead to the production of real-time PCR assays
was to combat the prolific spread of nosocomial pathogens. Namely,
carbapenem-resistant Enterobacteriaceae (CRE), particularly E.coli and K. pneumoniae,
research on CRE, multi-drug resistant
A. baumannii and P. aeruginosa, and methicillin-resistant
Staphylococcus aureus (MRSA) 9.

The goal of this review is
to  discuss
the application of real-time PCR in
Clinical microbiology with focus on microbial
genotyping, diagnosis, recent advances, microbiological
applications, limitation and advantages of PCR

 

 

 

 

Real-time PCR quantification

Specific Alleles and DNA are
detected and determined by the use of quantification
using real-time PCR.8 In
quantification, the number of nucleic
acids in a range of samples is detected and measured,
many of which end up impacting life science, agriculture or diagnostics.10 During quantification method,
the method of choice may vary depending on
the whether the quantification needs to be relative or absolute, the target
sequence, range of mRNA and the required accuracy11.

Based on the amount of template
within a sample the quantification can
either relative or absolute. In a
relative quantification, although the approach is simple it analyses changes in
a number of target sequences of gene expression in a given sample relative within the same matrix or
relative to a reference control11, 2. This approach examines the adequacy of the relative
expression ratio and the physiological changes within a gene expression11.  On the other hand, Absolute quantification
requires more effort. It is either based on an external
or internal cure, where known quantity is
used to quantitate the unknowns. This
approach is used when there is no standardized reagent or an absent of
sequential sequence2.

The use of real-time PCR for the quantification of the pathogen has
become increasingly popular for its use in pathogen diagnostic. Now, with the
availability of the amount of sequencing
data, designing micro-organisms assays has become easier8.
However, to do so, it is important that the necessary background steps and the information
are understood. To quantify a sample’s template, it is imperative that to contemplate the number of controls ant the type
of sample so that the concentration can
be acutely determined. Also, to prevent
competition within a wild-type template, it is important that the internal controls are added a suitable level. Since the internal controls are used to determine false-negatives during the quantification.
Finally, when expressing the results the to express the control to a suitable
biomarker of the organism2.

 

Quantification
using real-time PCR has many advantages. As mention, its speed,
sensitivity and specificity make it one
of the touchstones for nucleic quantification 10. Additionally, the technology can create a single reaction from the
multiplex amplification of several targets as well as a wide range of dynamic for
quantification (7–8 Log10). Although the multiplexing
option relies on the inclusion of
internal application it is essential for the diagnostic and detection of
each quantitative assay) 8. Nevertheless, the appearance
of current and improved formulae are
making quantification easier and more reliable2.

.

 

Microbial Genotyping

Constant
emphases in studies associated with diseases dealing with single nucleotide
polymorphisms (SNPs) has given rise to the genotyping platform and there is no
secret this has lead to a corresponding increase in methods used to investigate
diseases with SNPs association 12The identification of subtypes and variation of different pathogens at a
nucleic level is called genotyping . In Clinical microbiology genotyping plays
an important role in managing and the risk assessment of infectious diseases1

To characterize
unknown nucleic acid nucleotide sequencing is used. To reduce the lengthy
process real-time PCR became popular and
routine in declining deletion, or
insertions 2. Detection dyes such as Sybr
green and hybprob are the two most
commonly used in genotyping. Although other analyses such as light-up and double-stranded oligonucleotide and hyBeacons are not commonly used in this
function they could also function in the
same role2.

Moreover,
Numerous amount of probe-based genotyping
strategies for RT PCR has been established. In the presence of specific detection
probes, most of these methods contaiming PCR amplified DNA with polymorphism
sites can be be detected. More specifically alleles in a single tube
were recognized because of probes that were labelled with fluorophores13.  At endpoint analysis,
genotyped data are obtained after the
completion of PCR. Fluorophore and target
strand complexes formation are encouraged
when amplicon is denatured then rapidly cooled. 
Moderately, the temperature is
risen allowing the fluorescence from each vessel to be continuously recorded2.

Genotyping is quite useful, it can
be used in phylogenic analysis, in the
provision of extensive and accurate data once it’s
directly sequenced and it is also suitable for identical
of antimicrobial resistance and high-risk strains. Contrary, genotyping can be a very costly and laborious method1. Also, although genotyping
using real-time PCR can be quite useful when
it comes to microbial genotyping RT PCR is not the most effective method for
the analyzation of bacterial Strain. Since there is a limitation in genotyping of bacterial strains fact,  Repetitive
sequence-based PCR (rep-PCR) was developed to classify subspecies and strain of bacteria14.

In epidemiology study, the ultimate
resolution could be provided but if the entire genome of bacterial species is genotyped
but since only small regions o of the genome is interrogate the resolution is limited. An organism with high microbial genome makes it’s
it easier for healthcare centres to reconstruct
the pathway which could intern control the outbreaks.
A downside to this is that for each
pathogen an entire genome sequencing would have to be given to determine if it
is cost effeicient15.

 

Food and Safety in microbiology

Over the past 25 y, there have been
considerable advances in the

development and use of molecular
techniques for the detection of

microorganisms in foodstuffs as a
result of the increasing demand

for rapid results. These are normally
based on detecting specific

DNA or RNA target sequences using
amplification processes,

in particular the polymerase chain
reaction (PCR) (Cocolin and

others 2011). Their adoption, in many
instances, has replaced or

supplemented traditional culture
detection methods with culture

methods still recognized as the gold
standard for most bacterial

foodborne pathogens. But in the case of
some foodborne viruses,

which are not culturable, nucleic
acid-based assays remain the only

the choice
for their detection.

Annually, millions of people across
the world in both developed and undeveloped countries have died and are
currently dying or being infected by diseases caused by foodborne pathogens. Traditional culture methods were being been still being used in order to
detect pathogen transmitted through foods. This method of detection is
laborious and time-consuming16.

To overcome this dilemma, a rapid, cost-effective, and automated
diagnosis of food-borne was needed to reduce health hazard from food
contamination. This sparked the development first culture-independent molecular method around 18 years ago2, 5. This
allowed scientists to rely on the molecular
methods to study the micro-organisms in their environment. Now, there has been
more developed advances using molecular techniques to detect micro-organisms in
foods17. One of the techniques mostly
used to detect, identify and quantify foodborne pathogen is real-time PCR2.

Even though real-time PCR methods have replaced most traditional culture methods
the culture method is still useful and
are still used to detect most bacterial foodborne pathogens. Although some
foodborne pathogens such as bacteria can be detected by the use of culture,
other foodborne pathogens such as viruses cannot 17. Infections caused by
foodborne viruses is one of the world’s human dominating diseases.  In the Hepatitis B virus alone, approximately
350 are carriers and two billion people
being past carriers. The development of nucleic acid assay based methods such
as real-time PCR remains quite useful in the detection of viruses such as
Hepatitis B and others5. This is because use of
real-time PCR, the application process is
utilized in order to detect specific RNA or DNA sequences 17

Apart from Hepatitis B, there are
numerous other foodborne pathogens such as Mycotoxins are another major concern
in food contamination worldwide because of a negative
effect on human population 5.These infectious pathogens
only aided in the developmental use of PCR methods throughout the years.
Allowing guidelines to be established by ISO standards are used in the
quantification of foodborne pathogens2.

The use of real-time PCR has
numerous advantages in the use of food
microbiology. It is accurate in the diagnosis of diseases,
has a low carryover contamination, user-friendly
interpretation, and as suggested before
it is able to combine detection and amplification into one step5.

Despite the usefulness of real-time PCR has its limitations. For example,
quantification of bacteria in raw foods of
some viruses remains a difficult task. Also,
the technology has the inability differentiate
between live and dead cells and detects a
small number of bacteria. This inability
to pick up on these may result in
pathogenic bacteria ending up in processed
foods. In order to combat this problem pre- enriched agar could be used prior to real-time PCR to aid in its detection5. This will allow multiplication
of bacteria for detection.

 

Taxonomy and
Diagnosis

Microbial taxonomy seeks to accurately identify and monitor pathogens such as viruses, bacteria or parasites18. The importance of
identification is essential in
microbiology because it provides diagnosis
and treatment diseases in either, human animals and plants19.

In taxonomy the polyphasic approach
is being utilized in order to
collectively determine the position of an isoate
base on phenotypic and genotypic properties Also in recent years, sequences like 16S rRNA gene sequencing have been used for identification of microorganism and determining
their evolutionary relationship20,19.However, it was not until the early 90s that the use
of mathematics and computational studies determined that each genome has specific
-specific signatures. These signature are now what allow science to determine
the which species are dissimilar or related based on these signatures19

Approximately 70% of medical
decision is analysed in the lab, some of
which are clinical microbiology techniques are utilised. For example, sepsis culture-based
techniques remains a standard method of detection21. In the
United States, bloodstream infection
kills around 14-50 % of patients and now it is one
of the 11 leading causes of death18.  The lengthy
time of culture-based
method is inconvenient since most of the diagnosis and treatment are not be
given in the 6-hour window; thus,
mortality and morbidity increases. However, with the use of real-time
PCR results were able to be interpreted at a faster rate and reduced the amount
of time and the application was also able to tell the severity of the infection, an abundance
of each pathogen, and quantify polymicrobial pathogens to initiate a treatment for the infection20, 22.

However,
unlike viruses that might solely on the use of real-time PCR or other PCR based
assay viruses and fungi  use the
combination of culture-based methods and PCR based methods to come up with a
diagnosis and  the treatment of infections caused by these
pathogens with the exception of Chlamydia, methicillin-resistant Staphylococcus
aureus (MRSA), and drug-resistant Mycobacterium tuberculosis 15.

 

Other advantages and
Disadvantages

As mentioned before real-time PCR  reduced cross-contamination,
saves time, has a high sensitivity and specificity
throughout1, 23.Contrary to the advantage of real-time PCR there are many other
limitations. The technology has the inability if
the express gene is detected in a cell, tissue and the comparison among different
conditions cannot be compared24.  When compared to conventional PCR, real-time PCR has it has restricted multiples capabilities
and is very incompatible with some fluorescent
chemistries platform2.Also, the purification and synthesis of probes are very costly and difficult
to use13. However, in order to
increase the use of this technology in turnaround diagnosis and reduce the cost of real-time PCR affordably and 3D
manufacturing methods were invented25.

 

In conclusion, despite the
limitations of real-time PCR  it has revolutionalised
microbiology diagnostic and the way in we
view specimen. The PCR based
technology is still expanding and growing
at an exponential rate.

 

 

 

 

 

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