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POLYMERASE CHAIN REACTION

IN INFECTIOUS DISEASES

DR.T.V.RAO MD

BASICS

Dr.T.V.Rao MD

POLYMERASE CHAIN REACTION IN INFECTIOUS DISEASES DR.T.V.RAO MD BASICS Dr.T.V.Rao MD

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DR.KARY BANKS MULLIS

DISCOVERY OF PCR

Dr.Kary Banks Mullis received a Nobel Prize

in chemistry in 1993, for his invention of the polymerase chain

reaction (PCR). The

process, which Kary Mullis conceptualized in 1983, is hailed as one

of the monumental

scientific techniques of

the twentieth century.

DR.T.V.RAO MD

DR.KARY BANKS MULLIS DISCOVERY OF PCR • Dr.Kary Banks Mullis received a Nobel Prize in chemistry

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MOLECULAR MICROBIOLOGY

Molecular microbiology is

the branch of microbiology devoted to the study of the

molecular principles of the physiological processes involved in the life cycle of prokaryotic and eukaryotic microorganisms such as bacteria, viruses unicellular algae fungi, and protozoa. This includes gene expression and regulation, genetic transfer, the synthesis of macromolecules, sub-cellular

organization, cell to cell

communication, and molecular aspects of pathogenicity and

virulence.

DR.T.V.RAO MD

MOLECULAR MICROBIOLOGY • Molecular microbiology is the branch of microbiology devoted to the study of the

MOLECULAR BIOLOGY DEALS WITH

MOLECULAR BIOLOGY DEALS WITH • Molecular microbiology is primarily involved in the interactions between the various

Molecular microbiology is

primarily involved in the

interactions between the various cell systems of microorganisms including

the interrelationship of

DNA, RNA and protein

biosynthesis and the manner in which these interactions are regulated.

MOLECULAR METHODS ARE

REVOLUTIONIZING

The use of molecular biology techniques, such as nucleic acid

probing and amplification, provides the potential for

revolutionizing how we diagnose infecting pathogens and determining the relation between nosocomial isolates. In clinical microbiology, this means that we will be able to detect smaller

amounts of DNA or RNA of pathogens than is currently possible, that the time required to identify and determine the antimicrobial susceptibility of slow-growing pathogens will be dramatically reduced, and that the diagnosis of nonculturable organisms will become possible.

MOLECULAR METHODS IN DIAGNOSIS

The introduction of molecular methods will not only

depend on their performance for each individual

microorganism, but also on the clinical relevance of the

diagnostic question asked, the prevalence of the clinical problem and whether the new methods are

added to the procedures in use or will replace them. Therefore no general rules can be proposed, strategies have to be elaborated for each infectious agent or clinical syndrome.

WHY USE A MOLECULAR TEST TO

DIAGNOSE AN INFECTIOUS DISEASE?

Need an accurate and timely diagnosis

Important for initiating the proper treatment

Important for preventing the spread of a

contagious disease

WHY USE A MOLECULAR TEST TO DIAGNOSE AN INFECTIOUS DISEASE? • Need an accurate and timely

WHEN WE REALLY NEED MOLECULAR

METHODS ?

Molecular diagnosis is most appropriate

for infectious agents that are difficult to

detect, identify, or

test for susceptibility in a timely fashion

with conventional

methods.

WHEN WE REALLY NEED MOLECULAR METHODS ? • Molecular diagnosis is most appropriate for infectious agents

MOLECULAR BIOLOGY IS EMERGING IN

DIAGNOSTIC METHOD

Diagnostic microbiology is in the midst of a new era.

Rapid nucleic acid amplification and detection

technologies are quickly displacing the traditional assays based on pathogen phenotype rather than

genotype. The polymerase chain reaction (PCR) has

increasingly been described as the latest gold standard

for detecting some microbes, but such claims can only

be taken seriously when each newly described assay is suitably compared to its characterized predecessors

LEADING USES FOR NUCLEIC ACID BASED TESTS

Nonculturable agents

 

Human papilloma virus

Hepatitis B virus

Fastidious, slow-growing agents

 

Mycobacterium tuberculosis

Legionella pneumophila

Highly infectious agents that are dangerous to culture

Francisella tularensis

Brucella species

Coccidioidis immitis

LEADING USES FOR NUCLEIC ACID BASED TESTS • Nonculturable agents • Human papilloma virus • Hepatitis

LEADING USES FOR NUCLEIC ACID BASED

TESTS

In situ detection of infectious agents

Helicobacter pylori

Toxoplasma gondii

Agents present in low numbers

HIV in antibody negative

patients

CMV in transplanted organs

Organisms present in small volume specimens

Intra-ocular fluid

Forensic samples

LEADING USES FOR NUCLEIC ACID BASED TESTS • In situ detection of infectious agents • Helicobacter

MOLECULAR METHODS ARE NECESSARY IF THE TRADITIONAL METHODS PROVIDE POOR RESULTS ?

Microscopy gives false positive results - - T.vaginalis, N.gonorrhoeae

Intracellular pathogens

viruses, M.genitalium

Low sensitivity

Chlamydia sp.,Neisseria sp.

Seropositivity is common Chlamydia sp.

Subtyping is mandatory HSV, HPV, HCV

Microbial growth is slow M. tuberculosis

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

MOLECULAR DIAGNOSTICS HOW IT WORKS?

Every organism contains some unique,species specific DNA sequences

Molecular diagnostics makes

the species specific DNA visible

MOLECULAR DIAGNOSTICS HOW IT WORKS? • Every organism contains some unique ,species specific DNA sequences •

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

UNDERSTANDING THE BASIS OF

POLYMERASE CHAIN REACTION

UNDERSTANDING THE BASIS OF POLYMERASE CHAIN REACTION DR.T.V.RAO MD

DNA MOLECULE

DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine

DR.T.V.RAO MD

Adenine
Adenine
  • Thymine

DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
  • Guanine

DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
  • Cytosine

DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine
DNA MOLECULE DR.T.V.RAO MD Adenine Thymine Guanine Cytosine

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  • 1. DNA

  • 2. PCR

Targets

Denaturing

Primers

Annealing

Cycles

Requirements

OUTLINE

1. DNA 2. PCR • Targets • Denaturing • Primers • Annealing • Cycles • Requirements

DNA STRUCTURE

In double stranded linear DNA, 1 end of each strand

has a free 5’ carbon and phosphate and 1 end has a

free 3’ OH group.

The two strands are in the opposite orientation with respect to each other (antiparallel).

Adenines always base pair with thymine's (2 hydrogen bonds) and guanines always base pair with cytosine's (3 hydrogen bonds)

DNA

DNA is a nucleic acid that

is composed of two

complementary nucleotide building block chains.

The nucleotides are made up of a phosphate

group, a five carbon

sugar, and a nitrogen base.

DR.T.V.RAO MD

DNA DNA is a nucleic acid that is composed of two complementary nucleotide building block chains.

DNA

DNA has four nitrogen bases.

Two are purines ( 2 ringed base )

Adenine ( A ), Guanine ( G )

Two are pyrimidine's ( 1 ringed base )

Cytosine ( C ), Thymine ( T )

DNA DNA has four nitrogen bases. • Two are purines ( 2 ringed base ) •

DNA

These four bases are

linked in a repeated

pattern by hydrogen bonding between the nitrogen bases.

The linking of the two complementary strands

is called hybridization.

DNA These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen

DNA

A purine always links with a pyrimidine base to maintain the

structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds

between them.

Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds

between them.

DNA A purine always links with a pyrimidine base to maintain the structure of DNA. Adenine

DNA

Example of bonding pattern.

Primary strand

CCGAATGGGATGC

GGCTTACCCTACG

Complementary strand

DNA Example of bonding pattern. • Primary strand CC G AA T GGG A T G

Molecular diagnostics is a set of methods

to study primary structure (sequence) of DNA

Hybridization with complementary sequences

-A-A-T-T-C-G-C-G-A-T-G-

- T-T-A-A-G-C-G-C-T-A-C-

Amplification (synthesis) of species specific sequences PCR polymerase chain reaction

-A-A-T-T-C-G-C-G-A-T-G-

-A-A-T-T-C-G-C-G-A-T-G-

-A-A-T-T-C-G-C-G-A-T-G-

-A-A-T-T-C-G-C-G-A-T-G-

-A-A-T-T-C-G-C-G-A-T-G-

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

PCR

PCR is a

technique that

takes a specific sequence of DNA of small amounts and amplifies it to be used for further

testing.

PCR PCR is a technique that takes a specific sequence of DNA of small amounts and

PCR REQUIREMENTS

Magnesium chloride: .5-2.5mM

Buffer: pH 8.3-8.8

dNTPs: 20-200µM

Primers: 0.1-0.5µM

DNA Polymerase: 1-2.5 units

Target DNA: 1 µg

PCR REQUIREMENTS • Magnesium chloride: .5-2.5mM • Buffer: pH 8.3-8.8 • dNTPs: 20-200µM • Primers: 0.1-0.5µM

PCR TARGETS

The targets in

PCR are the

sequences of DNA on each end of the

region of interest, which can be a complete gene or small sequence.

PCR TARGETS The targets in PCR are the sequences of DNA on each end of the

PCR TARGETS

The number of bases in the

targets can vary.

TTAAGGCTCGA

. . .

.

AATTGGTTAA

The

. . . .

Represents the

middle DNA sequence,

and does not have to be

known to replicate it.

PCR TARGETS The number of bases in the targets can vary. TT AA GG C T

PCR DENATURING

Denaturing is the

first step in PCR,

in which the

DNA strands are

separated by heating to 95°C.

PCR DENATURING Denaturing is the first step in PCR, in which the DNA strands are separated

PCR PRIMERS

Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the

complementary building blocks of the target

sequence. Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence. A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both directions.

PCR PRIMERS Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the

PCR PRIMERS

TTAACGGCCTTAA

. . .

TTTAAACCGGTT

AATTGCCGGAATT

. . . . . . . . .

.>

and

<. . . . . . . . . .

AAATTTGGCCAA

TTAACGGCCTTAA

. . .

TTTAAACCGGTT

PCR PRIMERS

The primers are

added in excess

so they will bind to

the target DNA instead of the two strands binding back to each other.

PCR PRIMERS The primers are added in excess so they will bind to the target DNA

PCR ANNEALING

Annealing is the

process of allowing

two sequences of DNA to form

hydrogen bonds.

The annealing of the target sequences an

primers is done by

cooling the DNA to

55°C.

DR.T.V.RAO MD

PCR ANNEALING Annealing is the process of allowing two sequences of DNA to form hydrogen bonds.

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PCR TAQ DNA POLYMERASE

Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest.

Taq produces an enzyme

called DNA polymerase,

that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.

PCR TAQ DNA POLYMERASE Taq stands for Thermus aquaticus, which is a microbe found in 176°F

ESTABLISHMENT OF A PCR LABORATORY

To perform PCR for the repetitive detection of

a specific sequence, three distinct

laboratory areas are required. The specific technical operations, reagents ,and personnel considerations

ESTABLISHMENT OF A PCR LABORATORY • To perform PCR for the repetitive detection of a specific

PCR laboratory

DNA preparation

Sample handling

QC & QA Quality control & assurance No alternative
QC & QA
Quality control &
assurance
No alternative
  • Laboratory

PCR laboratory DNA preparation Sample handling QC & QA Quality control & assurance No alternative Laboratory
  • Mixing site

PCR laboratory DNA preparation Sample handling QC & QA Quality control & assurance No alternative Laboratory

Thermocycler

Amplification

PCR laboratory DNA preparation Sample handling QC & QA Quality control & assurance No alternative Laboratory
Detection
Detection

Documentation

Clean room

Stock solutions

R & D (Research and development)
R & D
(Research and development)

Alternatives: - commercial kits - robots + kits

PCR IN CLINICAL MICROBIOLOGY

Molecular detection has

mostly come to the

clinical microbiology

laboratory in the form of

PCR technology, initially

involving single round or

nested procedures with detection by gel electrophoresis.

PCR IN CLINICAL MICROBIOLOGY • Molecular detection has mostly come to the clinical microbiology laboratory in

HELPS RAPID DETECTION

TIMELY DIAGNOSIS CAN SAVE SEVERAL LIVES

HELPS RAPID DETECTION TIMELY DIAGNOSIS CAN SAVE SEVERAL LIVES • Polymerase chain reaction (PCR) techniques have

Polymerase chain

reaction (PCR)

techniques have led the way into this new era by allowing rapid detection of

microorganisms that were

previously difficult or impossible to detect by traditional microbiological

methods.

UNDERSTANDING THE PCR CYCLE

UNDERSTANDING THE PCR CYCLE DR.T.V.RAO MD

Isolation of Nucleic Acids

Goals:

removal of proteins DNA vs RNA isolation of a specific type of DNA (or RNA)

Types of DNA:

genomic (chromosomal)

organellar (satellite) plasmid (extra- chromosomal) phage/viral (ds or ss) complementary (mRNA)

Types of Methods:

differential solubility ‘adsorption’ methods density gradient centrifugation

General Features:

denaturing cell lysis (SDS, alkali, boiling, chaotropic) enzyme treatments

  • - protease

  • - RNase (DNase-free)

  • - DNase (RNase-free)

High MW Genomic DNA Isolation

Typical Procedure

  • 1 Cell Lysis

0.5% SDS + proteinase K (55 o several hours)

  • 2 Phenol Extraction

gentle rocking several hours

  • 3 Ethanol Precipitation

  • 4 RNAse followed by proteinase K

  • 5 Repeat Phenol Extrac-tion and EtOH ppt

EtOH Precipitation

2-2.5 volumes EtOH, -20 o high salt, pH 5-5.5 centrifuge or ‘spool’ out

High MW Genomic DNA Isolation Typical Procedure 1 Cell Lysis – 0.5% SDS + proteinase K

High MW Genomic DNA Isolation

Typical Procedure

  • 1 Cell Lysis

0.5% SDS + proteinase K (55 o several hours)

  • 2 Phenol Extraction

gentle rocking several hours

  • 3 Ethanol Precipitation

  • 4 RNAse followed by proteinase K

  • 5 Repeat phenol extrac-tion and EtOH ppt

Phenol Extraction

mix sample with equal volume of sat. phenol soln retain aqueous phase optional chloroform/isoamyl alcohol extraction(s)

High MW Genomic DNA Isolation Typical Procedure 1 Cell Lysis – 0.5% SDS + proteinase K

aqueous phase (nucleic acids)

phenol phase (proteins)

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PCR

The PCR reaction has three basic steps

Denature when you denature DNA, you separate it into single strands (SS).

In the PCR reaction, this is accomplished by heating at 95 0 C for 15 seconds to 1 minute.

The SS DNA generated will serve as templates for DNA synthesis.

Anneal to anneal is to come together through complementary base-pairing (hybridization).

During this stage in the PCR reaction the primers base-pair with their complementary sequences on the SS template DNA generated in the denaturation step of the reaction.

PCR

The primer concentration is in excess of the template concentration.

The excess primer concentration ensures that the chances of the primers base-pairing with their complementary sequences on the template DNA are higher than that of the complementary SS DNA templates base-pairing back together.

The annealing temperature used should ensure that annealing will occur only with DNA sequences that are completely complementary. WHY?

The annealing temperature depends upon the lengths and sequences of the primers. The longer the primers and the more Gs

and Cs in the sequence, the higher the annealing temperature. WHY? The annealing time is usually 15 seconds to 1 minute.

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DR.T.V.RAO MD

PCR CYCLES

PCR

Most PCR reaction use 25 to

30 of these cycles to amplify the target DNA up to a million times the starting

concentration.

PCR • Most PCR reaction use 25 to 30 of these cycles to amplify the target

PCR CYCLES

PCR

Extension during this stage of the PCR reaction, the DNA polymerase will use dNTPs to synthesize DNA complementary to the template DNA.

To do this DNA polymerase extends the primers that annealed in the annealling step of the reaction.

The temperature used is 72 0 C since this is the optimum reaction temperature for the thermostable polymerase that is used in PCR.

Why is a thermostable polymerase used? The extension time is usually 15 seconds to 1 minute.

The combination of denaturation, annealing, and

extension constitute 1 cycle in a PCR reaction.

PCR CYCLES

PCR CYCLES

PCR CYCLES

PCR CYCLES REVIEW

Denaturalization: 94°- 95°C

Primer Annealing: 55°-

65°C

Extension of DNA: 72°

Number of Cycles: 25-40

PCR CYCLES REVIEW • Denaturalization: 94°- 95°C • Primer Annealing: 55°- 65°C • Extension of DNA:

LEADING USES FOR NUCLEIC ACID BASED

TESTS

Differentiation of antigenically similar agents

May be important for detecting specific virus genotypes

 

associated with human cancers (Papilloma viruses)

Antiviral drug susceptibility testing

May be important in helping to decide anti-viral therapy to

 

use in HIV infections

Non-viable organisms

Organisms tied up in immune complexes

LEADING USES FOR NUCLEIC ACID

BASED TESTS

Molecular epidemiology

To identify point sources for hospital and community- based outbreaks

To predict virulence Culture confirmation

LEADING USES FOR NUCLEIC ACID BASED TESTS • Molecular epidemiology • To identify point sources for

APPLICATIONS OF PCR

The swab specimens can be stored 2-30°C for 4 days or frozen at -20°C.

The urine samples are refrigerated at 2-8°C or

stored at -20°C.

A target sequence is chosen for both, amplified with polymerase, and then

evaluated with an enzyme

immunoassay.

APPLICATIONS OF PCR The swab specimens can be stored 2-30°C for 4 days or frozen at

TARGET AMPLIFICATION

Target amplification

requires that the DNA to be

tested for be amplified, i.e., the number of copies of the DNA is increased.

To understand this we must

first review the activity of the enzyme, DNA polymerase, that is used to amplify the DNA.

TARGET AMPLIFICATION • Target amplification requires that the DNA to be tested for be amplified, i.e.,

POLYMERASE TEMPLATE AND PRIMER

REQUIREMENTS

DNA polymerase cannot initiate synthesis on its own.

It needs a primer to prime or start the

reaction.

The primer is a single stranded piece of DNA that is complementary to a unique region of the sequence to be amplified.

POLYMERASE TEMPLATE AND PRIMER REQUIREMENTS • DNA polymerase cannot initiate synthesis on its own. • It

PCR REACTIONS IN THE LAB

We will be doing two different PCR reactions in the lab.

For the first PCR reaction we will be using what are called consensus sequence primers.

These are primers that will bind to unique regions of the 16S ribosomal genes found in all bacteria.

The sequences of these primers are not unique to a specific kind of bacteria, but they are unique to a conserved region (consensus sequence) of DNA found in the 16S ribosomal genes of all bacteria.

They will be used to amplify a portion of the 16S ribosomal gene of an unknown bacteria.

PCR REACTIONS IN THE LAB

The sequence of the amplified DNA will be determined.

The identity of the unknown will be determined by searching the DNA sequence databases.

Note that that DNA of all bacteria should be amplified and yield a product using these consensus primers.

For the second PCR reaction we will be using primers that are unique to the genes that encode the shiga-like toxin produced by EHEC.

Only the DNA of those bacteria that carry the shiga-like toxin gene will be amplified and yield a product when using these primers.

For diagnostic purposes, only the second type of PCR, in

which primers unique to a single type of organism or gene

are used, is practical.

WHAT ARE THE ADVANTAGES OF USING

A MOLECULAR TEST?

High sensitivity

 

Can theoretically detect the presence of a single organism

High specificity

 

Can detect specific genotypes

Can determine drug resistance

Can predict virulence

Speed

 

Quicker than traditional culturing for certain organisms

APPLICATIONS OF PCR IN OPTIMAL

DIAGNOSIS IN INFECTIONS

Neisseria gonorrhea and

Chlamydia trachomatis are

two of the most common sexually transmitted diseases. The infections are asymptomatic and can

lead to pelvic inflammatory disease, salpingitis in women, epididymitis in men, infertility, and ectopic

pregnancy.

APPLICATIONS OF PCR IN OPTIMAL DIAGNOSIS IN INFECTIONS Neisseria gonorrhea and Chlamydia trachomatis are two of

Advantages

Molecular methods

High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration

In-house (home-brew) PCR methods

Cost effective High sensitivity

R&D is absolutely necessary

High quality Fast implementation of scientific discoveries Customer friendly

The 7th Baltic Congress in Laboratory Medicine, Pärnu 11.09.2004

WHAT ARE THE ADVANTAGES OF USING A

MOLECULAR TEST?

Simplicity

Some

assays

are now automated

WHAT ARE THE ADVANTAGES OF USING A MOLECULAR TEST? • Simplicity • Some assays are now

WHAT ARE THE DISADVANTAGES OF USING A

MOLECULAR TESTS ?

Expensive

So specific that must have good clinical data to support

infection by that organism before testing is initiated. Will miss new organisms unless sequencing is done as

we will be doing in the lab for our molecular unknowns

(not practical in a clinical setting).

May be a problem with mixed cultures would have to assay for all organisms causing the infection.

WHAT ARE THE DISADVANTAGES OF USING A

MOLECULAR TEST?

Too sensitive? Are the results clinically

relevant?

WHAT ARE THE DISADVANTAGES OF USING A MOLECULAR TEST? • T oo sensitive? Are the results

PCR TO RT PCR

Use of PCR in the field of molecular diagnostics has increased

to the point where it is now accepted as the standard method for

detecting nucleic acids from a number of sample and microbial types.

However, conventional PCR was already an essential tool in the

research laboratory. Real-time PCR has catalyzed wider

acceptance of PCR because it is more rapid, sensitive and

reproducible, while the risk of carryover contamination is minimized

OVERVIEW OF RT - PCR

tissue

OVERVIEW OF RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR

extract RNA

OVERVIEW OF RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR

copy into cDNA

(reverse transciptase)

OVERVIEW OF RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR

do real-time PCR

OVERVIEW OF RT - PCR tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR

analyze results

NEED FOR NOVEL METHODS IN DIAGNOSIS OF

INFECTIONS

Identification of the infectious agent(s) is essential to provide

an accurate diagnosis,

appropriately manage patient care and in certain cases reduce the risk of transmission within the

community and health care

settings. To meet these

challenges, innovative technologies have been developed that detect single pathogens, multiple syndrome

related pathogens and

genotypic drug resistance

NEED FOR NOVEL METHODS IN DIAGNOSIS OF INFECTIONS • Identification of the infectious agent(s) is essential

OUR VISION TO FUTURE DIAGNOSIS OF

INFECTIOUS DISEASES

With the ability to test for an unlimited number of potential pathogens simultaneously, next-generation sequencing has the potential to

revolutionize infectious diseases diagnostics

In the microbiology laboratory, this technology will likely replace the

traditional “one test, one bug” approach to pathogen diagnostics

The deep sequence information being generated is rapidly surpassing our capacity to analyze the data and will necessitate the development of highly parallel computational frameworks, such as cloud computing

In adapting this technology for clinical diagnostics, interpretation of data, appropriate quality control standards, and fulfilling regulatory requirements will be critical

One powerful application of next-generation sequencing is discovery of novel pathogens that may be associated with acute or chronic illnesses

Advantages

Molecular methods

High sensitivity and specificity Detects pathogen, not immune response Quick results High transport toleration

In-house (home-brew) PCR methods

Cost effective High sensitivity

R&D is absolutely necessary

High quality Fast implementation of scientific discoveries Customer friendly

MOLECULAR METHODS HAVE LIMITATIONS

However, because of their high specificity, molecular methods

will not detect newly emerging resistance mechanisms and are

unlikely to be useful in detecting resistance genes in species where the gene has not been observed previously. Furthermore, the presence of a resistance gene does not mean that the gene will be expressed, and the absence of a known resistance gene

does not exclude the possibility of resistance from another mechanism. Phenotypic antimicrobial susceptibility testing methods allow laboratories to test many organisms and detect newly emerging as well as established resistance patterns.

DIAGNOSTIC MICROBIOLOGY CHANGING FROM

PHENOTYPIC METHODS TO MOLECULAR METHODS

In hospital epidemiology, the use of such techniques has

already provided tests with exceptional discriminatory

power. Molecular techniques allow more efficient typing of

all pathogens, and permit discrimination between strains of organisms that were previously phenotypically identical or

uncharacterizable. Currently, cost and complexity limit the applicability of these techniques; however, they are likely to be developed for routine laboratory use in the next decade, and their impact will be considerable.

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