It is a presentation on an article about the development of PCRs to detect different types of Mycoplasma

1. Ximena Consuegra Ruiz
3rd Semester (2022-2)
Universidad Pontifica Bolivariana

2. Mycoplasma
Small bacteria with no cell wall
Contagious diseases
mastitis
respiratory conditions
reproductive conditions
arthritis
Types
M. bovis
M. arginini
M. bovigenitalium
M. californicum
M. alkalescens
M. dispar
M. canadens
About
Mycoplasma
bovis
|
Mycoplasma
bovis
[Internet].
mbovis.
2020
[cited
2022Aug.8].
Available
from:
https://www.mbovis.govt.nz/mycoplasma-bovis-
info-hub/whats-mycoplasma-bovis/
Mycoplasma:
All
You
Need
To
Know
[Internet].
STD.GOV
Blog.
2018
[cited
2022Aug.8].
Available
from:
https://www.std-
gov.org/blog/mycoplasma-all-you-need-to-know/
INTRODUCTION

3. PCR
Polymerase chain reaction
Amplify DNA (DNA polymerase)
Molecular and genetic analysis
Mapping techniques
DNA fingerprinting
Detection of bacteria or viruses
Diagnosis of genetic disorders
Conditions of protocol vary
1.
Sanskartutorials.in.
2022
[cited
2022
Aug
9].
Available
from:
https://sanskartutorials.in/wp-content/uploads/2021/04/what-is-
rt-pcr-test-why-take-rt-pcr-test-3.png?is-pending-load=1
INTRODUCTION

4. Develop a pair of polymerase chain
reaction primers for detecting
ruminant mycoplasma pathogens
GENERAL OBJECTIVE

5. DNA extraction from standard isolates
Culturing isolates in mycoplasma culture broth (at 37 °C for 72 h)
DNA extraction by boiling the isolates
Adjustment of the concentrations of the respective isolates
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MATERIALS AND METHODS
Sensitivity test using bulk and mastitis milk
samples
Collection of bulk milk and milk from cows with mastitis
Milk samples' (1 mL) centrifugation at 3,000 rpm for 10 min
Collection of 100 μL of the liquid supernatant
DNA from the milk samples was prepared (according to
AxyPrepTM Bacterial Genomic DNA Miniprep Kit (Corning
Inc.) protocol)
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3. SelectScience [Internet]. Selectscience.net.
2022 [cited 2022 Aug 9]. Available from:
https://www.selectscience.net/images/products/
258_axyprep_blood_dna_miniprep_medium.jpg.a
shx?width=270height=270bgcolor=ebebeb

6. Optimization of PCR forMycoplasmaspp.
Initial denaturation at 94 °C for 7 min
40 cycles of denaturation at 94 °C for 1 min
Annealing at 46 °C for 40 s
Extension at 72 °C for 1 min
Final extension step of 72 °C for 7 min
PCR products were separated through electrophoresis on 2% (w/v) agarose gels, stained with ethidium
bromide solution
Visualized with a UV transilluminator
Performed in a total reaction volume of 20 microliters
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Primer design
The chosen main sequence from the partial 16S rRNA sequence of M. Bovirhinis was
proven to be 90–99% homologous to M. Bovis'
Its most similar part to M. bovis was compared with the region with the highest
dissimilarity in other bacterial and animal genomes (U.S. National Library of Medicine,
BLAST®)
The oligonucleotide sequences (5’–3’) for the newly designed universal primers were
chosen.
The 16S rRNA gene sequences of various Mycoplasma species (NCBI’s Primer-BLAST tool)
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2.
3.
Size of amplicon: 233 bp
MATERIALS AND METHODS

7. RESULTS
DISCUSSION

8. RESULTS
DISCUSSION

9. Author Citation Agree / Disagree
Parker et al. 2018
A recent report described the limits
of detection for other PCR assays
Pfützner and Sachse 1996
the limit for detection using an ELISA
kit was as low as 1,000 cfu/mL in milk
after 48 h of incubation
McAulife et al. 2005
Some assays have more complex
requirements, such as denaturing
gradient gel electrophoresis (DGGE)
to differentiate species

10. Modern biological techniques, such as PCR, allow
faster and less complex diagnosis, which may
prevent further negative consequences.
1.
2. Well-managed biological tools lead to a better
understanding of small organisms such as Mycoplasma
and, thus, permit a more efficient way to detect them
and take an assertive course of action
CONCLUSIONS