For over 10 decades, agents of infectious diseases have been identified through their phenotype directly in specimen and after a growth in culture. Today, we are in a molecular era, there is an opportunity to detect organisms more rapidly and accurately based on their genetic signatures. Biomedical science research discovery offers a growing numbers of a nucleic acid amplification tests (NAATS) among which is polymerase chain reaction (PCR) for detection and identification of bacterial, parasitic, fungi and viral pathogens. These assays improve patient care, reduce antibiotic usage, enhance test utilization and increase laboratory and hospital efficiency. In this seminar, we will explore the clinical usefulness and potential of both conventional and real-time PCR assays in Clinical Microbiology.

1. APPLICATION OF PCR IN MICROBIOLOGY
A SEMINAR PRESENTED TO THE
DEPARTMENT OF MICROBIOLOGY,
FEDERAL TEACHING HOSPITAL,
ABAKALIKI (FETHA 1)
BY
NWUDELE CHIBUEZE DANIEL
29TH APRIL, 2016

2. MICROBIOLOGY AND PCR
• For over 10 decades, agents of infectious diseases have been identified
through their phenotype directly in specimen and after a growth in culture.
• Today, we are in a molecular era, there is an opportunity to detect organisms
more rapidly and accurately based on their genetic signatures.
• Biomedical science research discovery offers a growing numbers of a nucleic
acid amplification tests (NAATS) among which is polymerase chain reaction
(PCR) for detection and identification of bacterial, parasitic, fungi and viral
pathogens.
• These assays improve patient care, reduce antibiotic usage, enhance test
utilization and increase laboratory and hospital efficiency.
• In this seminar, we will explore the clinical usefulness and potential of both
conventional and real-time PCR assays in Clinical Microbiology.

3. There Are Several Reasons For The Use Of PCR
• Difficulties in identification of bacteria.
• Large time required for the identification with culture
techniques.
• The media required for identification and confirmation of
most pathogens are very expensive.
• A few bacteria in the environment are viable but not
culturable.

4. POLYMERASE CHAIN REACTION
•HAS MULTITUDE
OF
APPLICATIONS
IN
MICROBIOLOGY
4

5. Applications of PCR
Basic Research Applied Research
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies

6. APPLICATIONS OF PCR IN MICROBIOLOGY
• Clinical Bacteriology
• Clinical Parasitiology
• Clinical Virology
• Clinical Mycology
• Detection of Potential Bioterrorist Agents.
• Detection of Antimicrobial Agents.

8. CLINICAL BACTERIOLOGY.
• Detection of Bacterial Respiratory Pathogens.
• Detection of Bacterial Meningitis.
• Detection of Borrelia Species.
• Detection of Bacterial Gastrointestinal Pathogens, (David, 2012).

9. BACTERIAL RESPIRATORY PATHOGENS
• Bordetella Pertussis: slow growing fastidious organism and takes 3-12days
to be isolated by culture method.
• CLINICAL FINDINGS: Runny nose, sneezing, mild cough and low grade
fever.
• SPECIMEN: Nasopharyngeal aspirate, (0.5ml), (CDC, 2016). Optimal
timing for PCR has optimal sensitivity during the first 3 weeks of cough when
bacterial DNA is still in the nasopharynx. After the 4th week of the cough, the
amount of bacterial DNA rapidly diminishes, (CDC, 2015).
• PRINCIPLE: IS481 is an insertional sequence that is present in genome of
Bordetella pertussis , a causative agent of wooping cough in humans. Detection
of IS481 by PCR accurately identify B. pertussis and the procedure can be
invaluabe in the diagnosis of whooping cough (respiratory illness), (Reischl et
al, 2001).

10. BACTERIAL MENINGITIS
• Bacterial meningitis is a serious disease that affects the central nervous system
(CNS) causing significant morbidity and mortality.
• IMPLICATED PATHOGENS: Neisseria meningitidis, Streptococcus pneumonia
and Haemophilus influenza..
• CLINICAL FINDINGS: fever, headache and stiff neck.
• ACCEPTABLE SPECIMENS: CSF, Serum, or Blood, (mini volume, 0.25ml).
• INCUBATION PERIOD: 3-4days, with a range of 2-10days, (CDC, 2015).
• PRINCIPLE: This is based on the utilization of ctrA (N. meningitidis),
bexA (H. influenzae a-d,) and ply (S. pneumonia). Detection of any of the
gene material mentioned above in a clinical sample by real-time PCR assay is
an indication that the causative agent to bacterial meningitis is present,
(Corless,2001).

11. BACTERIAL GASTROINTESTINAL PATHOGENS
• Bacterial gastrointestinal pathogens include: Campylobacter jejuni, E. coli,
Salmonella, Shigella, staphylococcus, Yersinia, etc.
• The E. coli, 0157:H7 serotype has been responsible for both sporadic cases
and large number of outbreaks of infection including: cholecystitis,
bacteremia, urinary tract infection, etc. (Andrew, 2012).
• CLINICAL FINDINGS: Abdominal cramping, sudden watery diarrhea,
gas, loss of appetite, vomiting, fatigue, fever etc.
• INCUBATION PERIOD: 3-4days (ranges from 1-10days).
• SPECIMENS: stool (10g) and pure culture isolate.
• PRINCIPLE: E. coli type 0157:H7 is known to harbour the verotoxin 1 , 2
(VT1 , VT2) and eaeA genes. Presence of these genes in a clinical specimen
can be utilized in diagnosis, (Holland et al, 2000).

12. MYCOBACTERIUM TUBERCULOSIS
• Mycobacterium tuberculosis is the causative agent of tuberculosis.
• CLINICAL FINDINGS: coughing that lasts three or more weeks,
coughing up blood, chest pain, unexplained weight loss, fatigue, fever,
chills and night sweats, (CDC, 2015).
• INCUBATION PERIOD: 2-12 weeks.
• SPECIMENS: gastric lavage, sputum, serum and urine, (0.5-1ml).
• PRINCIPLE: IS6110 is an insertional sequence that is present in all
the genomes of all mycobacterium tuberculosis complex (MTC)
species, the causative agents of tuberclosis in humans. MTC consist of
the following subspecies: M.tuberculosis, M. bovis, M. africanum, M.
carneti & M, microti. The detection of IS6110 by PCR accurately
identifies MTC and the procedure can be invaluable help in the
diagnosis of TB, (David et al, 2006).

13. CLINICAL PARASITOLOGY
DETECTION OF ENTERIC PARASITES
• Enteric parasites include: Cyclosporida spp, Cyclospora cayetanesis, Entamoeba histolytica
• Cyclosporida spp example, C. parvum causes cryptosporidosis.
• CLINICAL FINDINGS: watery diarrhea, stomach cramps, dehydration, nausea,
vomiting, fever, weight loss.
• INCUBATION PERIOD: symptoms manifest within 2-10days of infection
(average 7days) after becoming infected with the parasite. Sample collection should be
made within the acute phase of the infection and process within 24hours of
collection, (CDC,2015).
• SPECIMEN: stool (0.5g or 0.5ml). Stool collected in absence of preservatives must
be refrigerated (4⁰C) or frozen. Preservatives include: Totalfix, Unifix, Ecofix and
modified PVA.
• PRINCIPLE: The principle is based on the use of 5ˈ-nuclease QPCR to detect
cp11 and 18SrRNA genes of C. parvum oocyst present in the sample, (Andrew,
2012).

14. DETECTION OF ENTERIC PARASITES CONTD.
• Cyclospora cayetanesis is a coccidian parasite that has recently
become regnized as an emerging pathogen of humans.
• C. cayetanesis causes prolonged diarrhoea, nausea, abdominal cramps,
anorexia and weight loss.
• Similarly to other enteric parasites, diagnosis dependent on faecal
microscopy which lack sensitivity and specificity.
• In PCR, 5ˈ-nuclease assay was described by Varma et al. (2003), this
targeted the 18SrRNA sequence of this organism and was
demonstrated to be senstive and specific for the detection of the
oocysts.

15. DETECTION OF ENTERIC PARASITES
• Entamoeba histolytica is an intestinal protozoan parasite which is endemic in
many parts of the world and is responsible for millions of cases of dysentary
and liver diasease per year, (Blessmann et al, 2002).
• CLINICAL FINDINGS: abscesses, infections, severe illness, death.
• INCUBATION PERIOD: this is commonly 2-4weeks but may range from
a few days to years, (CDC, 2016).
• SPECIMENS: stool, liver aspirate, (0.5g or 0.5ml).
• PRINCIPLE: small subunit,(SSU) rDNA gene is found to be specific for E.
histolytica cysts. Detecting of this gene by PCR in the patient’s extracted
sample is important is diagnosis of amoebiasis, (Moon et al, 2011).

16. DETECTION AND QUANTIFICATION OF MALARIA PARASITE
• Plasmodium spp: P. falciparum, P. vivax, P. ovale and P.malariae. P. falciparum is the
causative agent of malaria in Nigeria.
• CLINICAL FINDINGS: fever, chills, profuse sweating, headache, nausea,
vomiting, diarrhea, anaemia.
• INCUBATION PERIOD: 7-18days
• SPECIMENS: blood in EDTA (1ml) or filter papers. (NHS, 2015).
• PRINCIPLE: Use of real-time 5ˈ nuclease QPCR assay targeting the
18SrRNA gene of plasmodium falciparum. Presence of 18SrRNA in the
analyed specimen is utilized in the diagnosis of malaria, (David, 2012).

17. DETECTION OF TOXOPLASMIC GONDII
• This is an opportunistic pathogen that affects AIDS and
immunosuppressed individuals. It causes toxoplasmic encephalitis and
extracerebral toxoplasmosis which are serious life threatening diseases
mostly during pregnancy.
• Rapid diagnosis of infection in these two patient group is required to
allow timely initiation of treatment.
• Diagnosis is based on serological detection of specific anti-toxoplasma
immunoglobulin, or culture of aminiotic fluid or fetal blood which are
not always reliable indicators of active infection, (Andrew, 2012).
• Improvement in research has reordered successful use of PCR in the
diagnosis of this pathogen targeting 18SrRNA gene, with 100%
specificity, sensitivity and reliable,(Schulz et al, 2003).

18. CLINICAL VIROLOGY
• PCR methods have proven to be useful tools in the diagnosis and
management of a wide range of viral diseases, (Nester, 2002)
• Applications are utilized in:
• Detection of Respiratory Viruses.
• Detection of Herpes Viruses.
• Detection of Enterovirus Infections
• Detection of Hemorrhagic Fever Viruses.
• Viral Genome Quantification

19. WHAT YOU SHOULD KNOW ABOUT VIRAL SPECIMENS
• Virus is generally detectable by real-time PCR from 3 to 10days after symptoms
appear.
• Most viral specimens should be held at 2-8⁰C rather than frozen for short term
(<48hours) transit and storage. For delays exceeding 48hours, freeze viral specimen at
-70⁰C or below. Do not freeze at -20⁰C.
• Sterile body fluids like CSF do not require any transport medium and should not be
diluted.
• Only sterile Dacron or rayon swabs with plastic shafts or if available, flocked swabs
should be used.
• Calcium alginate swabs or swabs with wooden sticks should not be used as the may
contain substances that can inactivate some viruses and inhibit some molecular assays.
• Urine may be collected within 7days of symptom onset from every patient suspected
of respiratory disease outbreak.
• Stool may be collected within 14days of symptom onset from patient hospitalized as
part of respiratory suspected disease, (e.g.; SARS) for RT-PCR.

20. DETECTION OF RESPIRATORY VIRUSES
• Influenza viruses.
• Enteroviruses.
• Human metapneumovirus.
(Hmpv).
• Respiratory syncytial virus
(RSV).
• Severe acute respiratory
syndrome (SARS)
• Matrix protein A
• 5-non-coding region.
• N gene.
• N gene.
(Gueudin et al, 2003)

21. DETECTING INFLUENZAE VIRUS
• Influenzae virus is a common respiratory pathogen that is highly contagious and
responsible for considerable morbidity and mortality, (Andrew, 2012).
• CLINICAL FINDINGS: fever , muscle aches, headache, lack of energy, dry
cough, sore throat, nasal congestion, etc.
• Sample collection should be done within 4 days of symptoms manifestation.
• SPECIMENS: virus isolates, respiratory clinical specimens (i.e. nasopharyngeal
swabs, nasal swabs, throat swabs, nasal aspirates, nasal washes, lower respiratory
tract specimen). Minimum volume is 1ml, (CDC, 2016).
• PRINCIPLE: matrix protein A, MI matrix gene, hemaglutinin gene B.
• Van Elden et al. (2001). The assay simultaneously detects Influenza A and B
using a multiplex TaqMan and targets genes of the pathogen.

22. ENTREOVIRUS INFECTIONS
• Enterovirus (EV) are common causes of CNS infections including: viral meningitis and
encephalitis and are responsible for about 80% of viral meningitis cases.
• CLINICAL FINDINGS: mild symptoms include: runny nose, sneezing, cough, body and
muscle aches. Severe symptoms include: wheezing and difficulty breathing, (CDC, 2015).
• SPECIMENS: stool, serum. Throat or nsasl swab, CSF, vesicle fluid or lesion, rectal or
nasopharyngeal(NP)/oropharyngeal(OP) swabs.
• Collecting specimens during the first week of illness is ideal. Although the virus can be shed
in stool for several weeks. A specimen set collected in the second week of illness should
include a rectal swab or stool sample. Isolation of EV from CSF is most productive within
2-3days after onset of CNS manifestations.
• MINIMUM VOLUME: cell culture (0.5-1ml), CSF (60ᴜL), stool (10-20g), serum (2ml).
(CDC, 2016).
• PRINCIPLE: This is based on developing a 5ˈ nuclease/TaqMan real- time QPCR
assay utilizing a single tube RT end and amplification format. The assay targeted 5ˈ non –
coding region which is known to be specific for enterovirus, (David, 2012).

23. RESPIRATORY SYNCYTIAL VIRUS(RSV)
• RSV is the most common etiological agent of lower respiratory tract
disease in children. The virus has been classified into two subgroups, A &
B based on antigenic and genetic variation in structural proteins.
• CLINICAL FINDINGS: fever, prominent runny nose, nasal congestion.
• SPECIMENS: Upper or lower respiratory tract specimens, pure cultures
isolate. 0.25ml is the minimum volume, (CDC,2016).
• Hu et al. (2003) described a real-time QPCR using nasal aspirate and
targeted the conserved nucleocaspid (N) gene specific for detection and
sub-grouping of the A & B virus subtypes.
• The assay were demonstrated to be about 40% more sensitive than culture
and 10% more sensitive than IF, (Gueudin et al, 2003).

24. DETECTION OF HERPES VIRUSES
• The human herpes virus family include the clinically important pathogen herpes
simplex viruses 1 & 2 (HSV-1 & 2), varicella zoster virus (VZV) and human herpes
viruses 6, 7 8 (HHV-6, 7 & 8).
• CLINICAL FINDINGS: painful blisters or ulcer in or around the mouth, sore
on lip or cold sores, tingling, itching, burning sensation around mouth. Genital or
anal blisters for HSV-2.
• Sample collection is within the first 3 days from the date of onset of symptoms,
(WHO, 2016).
• Herpes simplex viruses and varicella zoster virus may not be recovered from lesion
beyond 5days after onset of clinical manifestation of disease, (CDC, 2016).
• SPECIMENS: CSF, saliva, whole blood, skin lesions. Minimum volume is 200ᴜL.
Skin lesions should be kept dry. Blood should be collected in EDTA or citrate
tubes. Heparin may cause interference with PCR and should be avoided, (CDC,
2016).
• Several gene targets have been selected for the detection of HSV DNA by real-
time PCR, including genes coding for glycoprotein B, C, D & G. thymidine kinase,
DNA polymerase and DNA binding protein. However, available reports so far
have utilize more DNA polymerase, (Espy, 2006).

25. VIRAL HAEMORRHAGIC FEVER
• Viral haemorrhagic fever (VHF) is a clinical syndrome caused by a number of
different viruses including Marburg virus (MBGV), Ebola virus (EBOV), Lassa virus
(LASV), Yellow fever virus (YFV), Dengue (DENV).
• CLINICAL MANIFESTATION of VHF infections include diarrhoea, myalgia,
cough, headache, pneumonia, encephalopathy and hepatitis. VHF can prove to be
fatal.
• INCUBATION PERIOD: Ebola 2-21 days while Lassa is 6- 21 days.
• Specimens: frozen tissue, blood & serum. Minimum volume, 1mL.
• 522-bp fragment of glycoprotein precursor gene spanning the entire RT-PCR
fragment(33nt) was amplified for sequence analysis of Lassa fever.
• In Ebola, significant amounts of non structural secreted glycoprotein (SGP) were
detected in actively infected humans, (Mehedi et al, 2011)

26. Diagnostic Assays [1]
• Ebola is a single stranded
RNA virus
• Genome is 18- 19 kb
in size
• The genome encodes for
7 viral proteins

27. Diagnostic Assays [2]
1
2
The NVRL performs two tests on each
sample. Each assay has a different
target.
1. Commercial real time Polymerase
Chain Reaction (PCR) assay
targeting the L (polymerase)
gene
1. In-house PCR assay targeting
the GP (glycoprotein) gene
The tests are run at the same time.

28. Packaging & Transport [1]
• Ebola is classified as a
Category A Infectious
Substances (UN2814) and
should be packaged in
accordance with international
regulations
• Transport to the NVRL via
the designated national
courier service for EVD
samples
©www.cdc.gov

29. Main corridor
BL3 laboratory
-50Pa
Class
I/ III
MSC
Class
III
MSC
Shower
-10Pa
Outer lobby
Inner lobby
-35Pa
-20oC -80oC
CO2
Inc.
40C
+10Pa
18 ac/h
Pass through
autoclave
Containment envelope
Testing Process [1]
On receipt in the NVRL the
sample is taken directly to
the Biosafety Level 3
Laboratory.

30. The specimen is unpacked,
prepared & undergoes
centrifugation.
RNA Extraction is performed. The
addition of Guanidine thiocyanate
inactivates the virus and PCR
analysis can be performed in the
Biosafety Level 2 Laboratory.
PCR analysis is performed.
Two assays with two separate
targets which are run in parallel.
B
S
L
3
B
S
L
2

31. VIRAL HAEMORRHAGIC FEVER
PRINCIPLE :
• Reverse transcription polymerase chain reaction, (RT-PCR) provides
a confirmatory diagnosis for Ebola virus. The principle of real-time
detection is based on the fluorogenic 5ˈ nuclease assay. During the
PCR reaction, the DNA polymerase cleaves the probe at 5ˈ end and
separate the reporter dye from the quencher only when the probe
hybridizes to the target DNA. This cleavage results in the fluorescent
signal generated by the cleaved reporter dye, which is monitored real-
time by the PCR detection system. The PCR cycle at which an
increase in the fluorescence signal is detected initially is proportional
to the amount of the specific PCR product. Monitoring the
fluorescence intensities in real –time allows the detection of the
accumulation product without having to re-open the reaction tube
after the amplification.

32. QUANTITATIVE VIRAL ASSAYS
• Quantitative viral assays have become
increasingly useful in the diagnosis and
management of viral diseases such as:
• Human Cytomegalovirus.
• Human immunodeficiency virus.
• Epstein Barr virus.
• Viral hepatitis agents.

33. HUMAN CYTOMEGALOVIRUS (HCMV)
• HCMV is the most common source of congenital malformation resulting
from viral intrauterine infection in developed countries, (Sumaira et al,
2011).
• CLINICAL FINDINGS: enlarged lymph nodes especially in the neck,
fever, fatigue, loss of appetite, malaise, muscle aches, rash, sore throat.
• SPECIMEN: urine, saliva, blood. Minimum volume, 5ml for urine, 200ᴜL
for serum and blood.
• At the present time, many real-time quantitative tests for CMV DNA have
been formatted on either the ABI or LC instruments. Choice of either
instrument depends on work flow issue in the laboratory rather than
specific performance characteristics of the platforms, (Espy et at, 2006).
• In either of the instruments, the following can be used: probe chemistry
(TaqMan), target genes (US17, UL83, UL123, Glycoprotein B),
(Espy et al, 2006).

34. HUMAN IMMUNODEFICIENCY VIRUS (HIV)
• The HIV-1 test is used as a monitor of the severity of the virus. The HIV-
1 causes a depletion of CD4+ T lymphocytes, causing immunodeficiency,
multiple opportunistic infections, malignancies, and death.
• The HIV-1 specimen is plasma(1ml) collected in EDTA that must be
separated from the cells within 6 hours.
• Heparin cannot be used as an anticoagulant because it inhibits PCR.
• A 142 base target sequence in the HIV-1 gag gene is converted from RNA
to complementary DNA, and to double stranded DNA using Thermus
thermophilus DNA polymerase in the presence of manganese and buffers,
which performs the reverse transcription and the amplification steps
simultaneously.
34

35. CLINICAL MYCOLOGY
ASPERGILLUS SPP
• Of all the fungal genera, Aspergillus has been of the most extensively targeted for
the development of real-time PCR assays, (Espy et al, 2006).
• The rationale behind this effort is that timely detection of Aspergillus spp. may
decrease the extreme morbidity and mortality associated with invasive aspergillosis,
(Espy et al, 2006).
• Currently, there are at least 167 recognized species and species variants of
Aspergillus, (http://www.ncbi.nih.gov/Taxonomy) but most cases of aspergillosis
are attributed to Aspergillus fumigatus, A. flavus, and A. niger,.
• Specimens which have been used in real-time PCR include: bronchoalveolar fluid
(BAL), blood, lung tissue, serum, cell pellet, culture isolate.
• TARGETED GENES: 18SrRNA, ITS, cytob, 58SrRNA, fks, 28SrRNA, mito. Trna.
• Report of application of PCR in the diagnosis of aspergillosis using bronchoalveolar
lavage fluid discovered the assay’s sensitivity to be 73% while specificity was reported
93%, (Espy et al, 2006).

36. CANDIDA SPP.
• The majority of real-time PCR assays developed to date for Candida
spp. have focused on the identification of the six or seven most
common species isolated from clinical specimens and most assays
analyzed isolates growing in pure culture, (Hsu et al, 2003; Lewis et al,
2003).
• Candida species are the fourth leading cause of nosocomial blood
stream infections and are associated with a majority rate of 40 to 50%
so rapid and reliable detection of candidemia has attracted significant
interest, ( Pappas et al, 2003; Gudlugsson et al, 2003).
• Blood and culture isolates have been used as specimen to diagnose this
disease.
• Targeted genes include 18SrRNA, act, erg11, ITS, ITS2, cdr.
• Candida infections has been evaluated using PCR aassay method and
the sensitivity and specificity for Candida albicans detection was
reported as 100% and 97% respectively, (Espy et al, 2006).

37. Potential Agents Of Bioterrorism
Bacillus anthracis.
• Speccimen: bacterial isolates.
• Target genes: capB, pagA, rpoB, capC genes.
Yersinia pesti.
Specimen: bacterial isolates
Target genes: 16SrRNA, pla, capI, ymt genes.

38. Detection Of Antimicrobial Resistance
• Antimicrobial resistance is resistance of microorganism to an antimicrobial
drug that was originally effective for treatment of infectious caused by it.
• Resistance microorganisms (including bacteria, fungi, viruses, and
parasites) are able to withstand attack by antimicrobial drugs, such as
antibacterial drugs (e.g. antibiotics), antifungals, antivirals and antimalarias
so the standard treatment become ineffective and infections persist,
increasing the risk of spread of others.
• Examples include: methicillin –resistant staphylococcus aureus (MRSA),
plasmodium falcipaurum multidrug resistance, multidrug-resistance
tuberculosis (MDR-TB), etc.
• In MDR-TB, 16SrRNA and a region of heat shock protein 65 gene
(hsp65) for DNA sequencing-based confirmation was utilized.
• In MRSA, mecA is targeted for amplification and diagnosis of the
pathogen.

39. Extraction of DNA from Urine
• Add 1mL of urine in 1.5mL mircrocentrifuge tube and spin for 3minutes
at 14000rpm.
• Aspirate the supernatant to the 0.1mL mark and discard.
• Re-suspend the pellet in 1mL of PBS/1% saponin solution by gentle
tapping and vortexing and allow for 20mins at room temperature to lyse.
• Following lysis, spin the sample material again for 2mins at 14000rpm,
keep the sediment and discard the supernatant.
• Re-suspend the sediment in 1mL of PBS and spin again for 2mins at
14000rpm.
• Discard the supernatant and add 100uL suspension of 20% chelex in
autoclaved PCR grade water to sediment and mix thoroughly.
• Boil the mixture for 13mins and spin for 3mins at 14000rpm.
• Carefully transfer the resultant which contain the DNA into a pre 1.5mL
microcentrifuge, excluding chelex for immediate PCR or store at -20⁰C

40. Why Must We Be Familiar With Molecular Methods?
• In many developed countries several diagnostic methods
are switched on to molecular methods.
• No scientific journal is willing to accept or publish any
article without incorporation of molecular methods.
• Antibiotic drug resistance is a growing concern to the
world, unless molecular identification is performed on
responsible genetic mechanisms , no effective scientific
conclusions can be drawn to contain the spread.

41. LIMITATIONS
• Despite the numerous advantages and the widespread acceptance,
the use of nucleic acid amplification techniques like PCR have
revealed several potential shortcomings.
• Standardized procedures for amplification methods are not yet
widely available.
• Dramatic inter laboratory variability in test results using the same
methods is not uncommon.
• Amplicon contamination in PCR laboratories continues to pose a
significant problem.
• Due to the high sensitivity of these assays, low levels of clinically
insignificant pathogens may be detected and mislead clinicians.
• Cost of the equipment may not be affordable especially in poor
resource setting.
• Complexicity of operation, requiring highly trained personnel to
generate accurate result.

42. CONCLUSION
• Based on the discoveries made during this study which is contained in the
discussion above, the following could be concluded:
• Methods associated to molecular biology have made excellent progress,
with clear usefulness in diverse fields of medical microbiology.
• The discovery of polymerase chain reaction (PCR) introduced a
technological advancement that is relevant for the detection of
microorganism, increasing the sensitivity, precision and accuracy of the
diagnosis.
• However, based on the limitations discovered during this study, it is
essential for laboratories to participate actively in interlaboratory quality
control programmes and communicate with each other to address
problems like standardization and optimization of PCR assays on a
continual basis