Pharmaceutical biotechnology

1. MOLECULAR & ENVIRONMENTAL
MICROBIOLOGY
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2. MOLECULAR MICROBIOLOGY
 “Molecular microbiology is the branch of microbiology devoted to the study of the
molecular basis of the physiological processes that occur in microorganisms, and the
manipulation of DNA”.
 Molecular microbiology is the fastest growing discipline which plays significant role
for the detection and characterization of microorganism.
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3. IMPORTANCE OF MOLECULAR MICROBIOLOGY
 Molecular techniques are major tools for the analysis of microorganisms(Begum and
Begum 2011)
 The introduction of these molecular techniques and their automation was introduced
in the form of PCR technology. And these automation when applied to various stages
of DNA or RNA extraction, a further increase the utility of molecular detection in the
clinical microbiology laboratory by making it more efficient and cost-effective.
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4. MOLECULAR MICROBIOLOGY TECHNIQUES
 Polymerase Chain Reaction (PCR)
-RT-PCR (Reverse transcriptase- PCR)
-Quantitative real time PCR(Q-RT PCR)
-Nested PCR
-Multiplex PCR
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5. POLYMERASE CHAIN REACTION (PCR)
 PCR targets and amplifies a specific region of a DNA strand. It is an in-vitro
technique to generate large quantities of a specified DNA.
 PCR can enzymatically amplify minute quantities of DNA or RNA to large number of
copies in a short period hence provides ample target that can be readily detected
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6. POLYMERASE CHAIN REACTION (CONT..)
 The basic steps in PCR are:
 Denaturation of DNA to single strands (94° C or higher for 15 seconds to 2 minutes)
 Annealing of primers to DNA (55° C, 1-2 mins)
 Extension by thermostable DNA polymerase (72° C typically 10-15 minutes).
 The same cycle is repeated for 30-35 times to produce approx. 109 copies / 2-3 hrs.
 All the steps of PCR are performed on the reaction mixture consisting of target DNA,
primer pairs, thermostable DNA polymerase (Taq Polymerase), deoxynucleotides
(dATP, dTTP, dGTP and dCTP), buffer and Mg salt in the same test tube.
 Thermocyclers are used for the process(Singh et al., 2023).
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7. POLYMERASE CHAIN REACTION (CONT..)
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Fig 1: Polymerase chain reaction

8. POLYMERASE CHAIN REACTION (CONT..)
 PCR have following common types:
 RT-PCR (Reverse transcriptase- PCR):
 Used to detect viral nucleic acid (of RNA viruses) in clinical specimen.
 Using the enzyme reverse transcriptase, a complimentary copy of the RNA is made.
 A primer is annealed to the template RNA and with the help of reverse transcriptase.
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9. POLYMERASE CHAIN REACTION (CONT..)
 Quantitative real time PCR(Q-RT PCR)
 It quantitatively measures starting amounts of DNA, cDNA or RNA
 It is commonly used to determine whether a DNA sequence is present in a sample and
the number of its copies in the sample
 Detection is based on fluorescence technology using fluorescent dyes, such as Sybr
Green, to measure the amount of amplified product in real time
 The marker added to the sample and the signal is amplified with the amplification of
copy number of sample DNA(Espy, Uhl et al. 2006)
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10. POLYMERASE CHAIN REACTION (CONT..)
 Multiplex PCR:
 In multiplex PCR, two or more unique target sequences can be amplified
simultaneously.
 Multiplex PCR can also be used to test for different organism on a single
specimen(Clark et al., 2023).
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11. POLYMERASE CHAIN REACTION (CONT..)
 Advantages of PCR:
 It is a simple technique to understand and to use, and it produces results rapidly.
 It is a highly sensitive technique with the potential to produce millions to billions of
copies of a specific product for sequencing, cloning, and analysis.
 This is true of qRT-PCR as well, but qRT-PCR has the advantage of quantification of
the synthesized product. Thus, it can be used to analyze alterations of gene expression
levels in tumors, microbes, or other disease states.
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12. POLYMERASE CHAIN REACTION (CONT..)
Limitations of PCR:
 Although PCR is a valuable technique, it does have limitations. Because PCR is a
highly sensitive technique, any form of contamination of the sample by even trace
amounts of DNA can produce misleading results.
 In addition, in order to design primers for PCR, some prior sequence data is needed.
Therefore, PCR can only be used to identify the presence or absence of a known
pathogen or gene.
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13. MAJOR APPLICATIONS OF MOLECULAR MICROBIOLOGY
 Detection of Human Papillomavirus (Diagnosis of non-cultural agents: e.g. Human
papilloma virus, Hepatitis B virus etc.)
 For the purpose of molecular epidemiology e.g. identify point sources for hospital and
community-based outbreaks (Andrade, Bispo et al. 2008)
 They allow the fabrication of disposable single-use cartridges, resulting in rapid and
affordable diagnostics without the need for sophisticated and expensive laboratory
equipment.
 Presently, LOCs are being developed to test sepsis endocarditis, HIV, tuberculosis,
severe acute respiratory syndrome (SARS), pneumonia, malaria, and numerous other
infectious diseases(Basu, Seggerson et al. 2004).
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14. 14
Environmental microbiology is the study of the relationships between
microorganisms and the environment, including the composition, structure and
physiology of microbial communities.
The environment not only consists of the soil, water, air, sediment, and rocks
covering the planet, but also includes the animals and plants that inhabit these
areas.
ENVIRONMENTAL MICROBIOLOGY

15. ENVIRONMENTAL MICROBIOLOGY
Enivironmental
Microbiology
Industrial
Microbiology
Food safety
Health/infection
control
Aeromicrobiology
Aquatic
microbiology
Soil
microbiology
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16. BACKGROUND
In 1970, Several
events occurred
simultaneously
that highlighted
the need for a
better
understanding of
environmental
microorganisms.
The discovery of
the structure of
DNA in 1953 by
James Watson and
Francis Crick
engendered the
development of
new technologies,
notably the
polymerase chain
reaction (PCR),
based on nucleic
acids for
measuring and
analyzing
Simultaneous
impact of these
events caused
scientists to
question the
notion that our
food and water
supplies are safe
and also allowed
the development
of tools to increase
our ability to
detect microbes
and their activities
in environment.
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17. Symbiosis refers to the close
relationship amongst two
different organisms or living
things belonging to different
species.
EXAMPLE:-Lactobacillus and
humans, cells and mitochondria,
ants and fungi, coral and algae,
and cleaner fish are some
examples of symbiosis.
SYMBIOTIC MICROBES:
One organism lives inside another — the
host. Microbial symbiosis occurs between
two microbes. Microbes, form associations
with other types of organisms, including
plants and animals.
HOST- usually the LARGER member.
SYMBIONT- usually the SMALLER
member
SYMBIOSIS
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18. TYPESOF SYMBIOSIS
MUTUALISM
• Both the involved organisms benefit from each other. Example – Coral reefs are
nothing but a mutualistic association between coral organisms and various types
of algae residing inside them.
COMMENSALI
SM
• Only one organism benefits, while the other is neither benefited nor
harmed. Example – Hermit crabs use gastropod shells to protect their bodies.
PARASITIS
M
• One organism is benefited while the other organism is harmed. Example – Liver
fluke attaches itself to the liver and makes its way to the tissue and bile.
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19. RECENTLY DISCOVERED MICROBES
Agent Type of microorganism Mode of transmission Disease/symptoms
SARS (a coronavirus) Virus Fomites Respiratory
Escherichia
coli O157:H7
Bacterium Foodborne, waterborne
Enterohemorrhagic
fever, kidney failure
Hepatitis E virus Virus Waterborne, foodborne Hepatitis
Bird flu (avian influenza
A (H5N1))
Virus
Aerosol, fomites,
foodborne
Respiratory
Norovirus Virus Waterborne, foodborne Diarrhea
Helicobacter pylori Bacterium Foodborne, waterborne Stomach ulcers
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20. RECENTLY DISCOVERED MICROBES
Agent
Type of
microorganism
Mode of
transmission
Disease/symptoms
Cyclospora Protozoan
Foodborne,
waterborne
Diarrhea
MRSA (antibiotic-
resistant Staphyloc
occus aureus)
Bacterium Fomites Severe skin infections
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21. In modern environmental microbiology, pathogens and bioremediation remain fundamental to the
field, but in both cases these subject areas have been greatly enhanced through the application of
molecular genetics and biotechnology tools.
Vaccine development is often not rapid enough to guarantee protection and so the
environmental microbiologist must be prepared to develop better methods to reduce or stop
the spread of these agents throughout the environment.
New chemicals have been detected in ground and surface waters, air and soil that serve as
our potable resources. In addition, our ability to detect chemicals has gotten better and more
comprehensive.
MODERNENVIRONMENTALMICROBIOLOGY
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22. REFERENCES
 Begum, A., & Begum, S. (2011). Role of Molecular Biology as Diagnostic tools of Infectious disease-an update. Bangladesh Journal of Medical
Microbiology, 5(2), 27-29.
 Singh, Y., Anand, K. J., Singh, M., Ambawat, S., & Tiwari, G. (2023). Recent Developments in PCR Technology. BIOLOGICAL SCIENCES, 77, 79.
 Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., ... & Smith, T. (2006). Real-time PCR in clinical microbiology: applications
for routine laboratory testing. Clinical microbiology reviews, 19(1), 165-256.
 Clark, T. W., Lindsley, K., Wigmosta, T. B., Bhagat, A., Hemmert, R. B., Uyei, J., & Timbrook, T. T. (2023). Rapid multiplex PCR for respiratory viruses
reduces time to result and improves clinical care: Results of a systematic review and meta-analysis. Journal of Infection.
 Malamud, D., Bau, H., Niedbala, S., & Corstjens, P. (2005). Point detection of pathogens in oral samples. Advances in dental research, 18(1), 12-16.
 Liao, J. C., Mastali, M., Gau, V., Suchard, M. A., Møller, A. K., Bruckner, D. A., ... & Haake, D. A. (2006). Use of electrochemical DNA biosensors for rapid molecular
identification of uropathogens in clinical urine specimens. Journal of clinical microbiology, 44(2), 561-570.
 Basu, M., Seggerson, S., Henshaw, J., Jiang, J., del A Cordona, R., Lefave, C., ... & Basu, S. (2004). Nano-biosensor development for bacterial detection
during human kidney infection: use of glycoconjugate-specific antibody-bound gold NanoWire arrays (GNWA). Glycoconjugate Journal, 21, 487-496.
 Andrade, S. S., Bispo, P. J., & Gales, A. C. (2008). Advances in the microbiological diagnosis of sepsis. Shock, 30(7), 41-46.
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