Mycoplasma species detection by
PCR
Test
131 – Mycoplasma species Multiplex Detection by Polymerase Chain
Reaction
FAQs:
Q.
What is the best way to find out if my cells or biological materials are
contaminated with Mycoplasma species?
A.
The quickest and most sensitive method to detect Mycoplasma species
contamination is detection by Multiplex Polymerase Chain Reaction, a test that
can detect most known species of Mycoplasma, Acholeplasma and Ureaplasma.
Q.
How much sample do I need to order the Mycoplasma detection test?
A.
The test requires 0.5 ml of spent medium if you are testing cells in culture
(medium that had been on the cells for three or more days with no
antibiotics). You can also send a frozen vial of cells at room
temperature or ice packs. If you have a smaller volume, that would be
fine and we will adjust the volume to what the DNA extraction protocol
suggests.
Q.
Do I have to send my sample for Mycoplasma detection on dry ice?
A.
No. Samples can be shipped at room temperature or on ice packs.
Unless you are requested Mycoplasma elimination (Protocol CB124 – which
removes the Mycoplasma contamination from your cell line using an
antibiotic cocktail known to effective in eliminating Mycoplasma contamination),
you do not need to ship on dry ice. DNA is stable at room temperature.
Q.
What is the turnaround time for the Mycoplasma testing / detection
assay?
A.
Turnaround time is 24-72 hours. You would receive a PDF copy of the
report and a hard copy follows in the mail.
Q.
What is the cost for the test per sample for Mycoplasma testing?
A.
The cost is $109 if it is a biological sample and $125 if it is a patient
specimen (blood, tissue, secretions, etc). There is a volume
discount. If you ship more than 20 samples at a time, the price drops to
$99 per sample.
Q.
What is the report format for Mycoplasma testing?
A.
You will receive a three page report that lists the sponsor’s information,
materials and methods, gel image electronically labeled with two Mycoplasma
positive controls, one negative control and the samples. The final result
is listed clearly on the last page.
Q.
Do I need to notify the laboratory before shipping?
A.
No. The assay is performed 5 days a week and there is no need to inform
that lab.
Q.
What species of Mycoplasma are detected?
A.
we counted 25 of the most common species of Mycoplasma, Acholeplasma
and Ureaplasma based on actual sequence alignments on Genbank’s
published 16S Mollicute sequences.
Introduction
and Background:
Mycoplasma
refers to a genus of bacteria that lack a cell wall with more than 120 species.
Mycoplasma is the smallest known cell and is about 0.1 micron (μm) in
diameter. Mycoplasmas generally possess a relatively small genome
of 0.58-1.38 megabases, which results in drastically reduced biosynthetic
capabilities and explains their dependence on a host. Mycoplasmas
depend on sterols for the stability of their cytoplasmic membrane and they
obtain sterols from the host’s cholesterol. They are known to have a low
GC content in their genomes (23-40%).
The
lack of a cell wall offers them drug resistance to commonly used antibiotics
that inhibit cell wall synthesis such as penicillin or other beta-lactam
antibiotics. Mycoplasma species have adapted over time to live as independent
bacteria or parasitize the host. They have a wide host range that allows
them to infect humans, animals, plants and other genera. While many
species of Mycoplasma are considered commensals and are able to live in
the host without causing disease or can be considered opportunistic in
immunocompromised hosts, others are implicated in clinical conditions.
Atypical or walking pneumonia is often caused by Mycoplasma pneumoniae
or Mycoplasma hominis while non-specific urethritis is very commonly
caused by Mycoplasma genitalium which is also thought to be involved in
pelvic inflammatory disease. Several species are pathogenic
in humans, including M. pneumoniae, which is an important
cause of atypical pneumonia and other respiratory
disorders, and M. genitalium, which is believed to be
involved in pelvic inflammatory diseases.
Mollicutes
have been reported to seriously interfere with life sciences research when cell
lines or laboratory animals are infected. The bacteria can live
inside and outside of the cells, deplete nutrients from the medium and change
the biology of cells, disrupt metabolism, modify the immune response, inhibit
or prevent viral replication or have unexplained findings in cell culture
experiments.
Nocard
and Roux reported the cultivation of the causative agent of Contagious Bovine
Pleuro Pneumoniae (CBPP) in 1898, a disease that was widespread in cattle herds
and was found to be caused by M. mycoides subsp. mycoides SC
(small-colony type). In 1956, the first species of Mycoplasma were
isolated in culture. The most commonly encountered species are Acholeplasma
laidlawii, Mycoplasma arginini, M. orale, M. salivarium, M. fermentans, and
M. hyorhinis. Species such as Ureaplasma urealyticum, M.
pneumoniae and M. pirum are rarely present in cell cultures.
It is anticipated that many of the cell lines created prior to 1990 were
infected by Mycoplasma species that originated from contaminated animal
serum and further disseminated by contaminated aerosols in the laboratory and
laboratory technicians while they conducted their experiments.
A.
laidlawii and M. arginini are of bovine origin; M. orale and M.
salivarium are of a human origin (oropharynx) and M. hyorhinis
originated from swine. It is hypothesized that presence of Mycoplasmas
of swine origin in bovine serum is justified by the contamination of this
product in mixed slaughterhouses.
It
is estimated that as many as 30% of all cell lines generated prior to 1990 are
contaminated with Mycoplasma species.
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