Multiplex PCR method for

detection of bacterial
contamination in Food

KIRUBHA PAULDAS
MSc -II, SEM-III
ROLL NO:- 04

Large-volume immunomagnetic separation

combined with multiplex PCR assay for
simultaneous detection of Listeria
monocytogenes and Listeria ivanovii in
lettuce

Yan Mao, Xiaolin Huang, Sicheng Xiong , Hengyi

Xu .
http://dx.doi.org/10.1016/j.foodcont.2015.06.048
Food Control - Vol 59 (2016) 601-608

Listeriosis is afoodborne
illnesscaused by Listeria
monocytogenes.
Symptoms include fever and
chills, headache, upset stomach
and vomiting.
Treatment is with antibiotics.

Preparation of anti-Listeria mAb-coated MNBs

Suspended in sterilized PBS

containing 0.2% NaN3, and
stored at 4C

Procedure of large-volume IMS

live Listeria
monocytogenens

mAb-MNBs
in St. PBS

or L. ivanovii
concentrations ranging
from 10^1 CFU/mL to
10^8 CFU/mL

where Nu is the number of

unbound bacteria in the
supernatant (CFU/mL), and Nb
is the number of cells bound to
the mAb-MNBs (CFU/mL).

Incubated at RT for 90
min in shaker and then
separated using
external Magnetic field
and re-suspended in
PBS

mAb-MNB-bacterial pellets were

spread onto selective PALCAM
plates, for bacterial enumeration
after dilution, and each sample was
plated six times

DNA
extraction
mAb-MNB-bacterial pellets was heated in BWB for 10 min to detach the
target bacteria from the mAb-MNBs.
The mAb-MNBs were separated by an external magnetic field, and the
supernatant solution was centrifuged at 12,000 rpm for 3 min.
The bacterial pellets were resuspended in lysis buffer containing
lysozyme Na2EDTA ,Tris and Triton and incubated at 37C for 30 min.
Proteinase K (20 mg/mL) and GL buffer were added, and the mixture
was incubated at 57C for another 30 min.
After treatment with an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1), the mixture was centrifuged and ammonium
acetate (7.5 mol/L), glycogen (20 mg/mL), and ethanol were added to
the supernatent.
The mixture was cooled on an ice batch for 30 min, and centrifuged
at 4 C for 30 min. Lastly, the DNA pellet was dissolved in sterile pure
water for mPCR assays.

mPCR assay
Primers- Amplification of actA and iactA were designed to identify L.
monocytogenes
and L. ivanovii
Internal amplification control (IAC)
Listeria primer
Reaction Mixture- 10 X PCR buffer
0.26 mM dNTP
2.8 mM MgCl2
2.5 U Taq DNA polymerase
target DNA template
E. coli DNA template (for IAC),
specific primers
PCR conditions- 95 C for 2 min
35 cycles of 94 C for 30 s
52 C for 30 s
72 C for 30 s
72 C for 10 min

DNA
Denaturation
Primer
annealing
Elongation

The PCR products were electrophoresed on 2.5% agarose gel using 1

Detection of L.
monocytogenes and
L. ivanovii in spiked lettuce
samples

1 g of lettuce
was added
into 9 mL of
sterilized
PBS, and
homogenized
for 10 min to
produce 1:10
lettuce
homogenate.

The
homogenate
was spiked
with L.
monocytogene
s and L.
ivanovii at a
final
concentration
of 10^1 CFU/g
to10^8 CFU/g.

700 mg of
mAb-MNBs
was added to
10 mL of the
spiked
homogenate
and then
incubated at
room
temperature
for 90 min
with shaking.

After
separation
with an
external
magnetic field
for 5 min, the
mAb-MNBbacterial
complexes
were resuspended in
100 mL of
sterilized PBS.
The target
DNA was
isolated, and
mPCR analysis
was executed.

RESULTS AND DISCUSSION

Optimization of the amounts of SA and biotin-mAbs
large-volume IMS

Characterization of large-volume IMS

mPCR-Specificity of primers :-

Detection of L. monocytogenes and L.

ivanovii in lettuce using IMS-mPCR

Conclusion
mPCR assay coupled with large-volume (10 mL) IMS for the simultaneous
ultrasensitive detection of L. monocytogenes and L. ivanovii in lettuce
without an additional pre-enrichment period was succesful.
Under the optimal conditions, the CE of large-volume IMS for L.
monocytogenes and L. ivanovii was greater than 80% when the bacterial
concentration was less than 10^5 CFU/mL in real lettuce samples.
The LOD of IMS combined with mPCR assay for L. monocytogenes and L.
ivanovii reached as low as 1 CFU/mL in pure culture and 10 CFU/g in spiked
lettuce samples.
Moreover, the proposed IMS-mPCR method also showed excellent
discrimination of other common non-target pathogens, and the total assay
time took only less than 7 h, including sample preparation, large-volume IMS,
and mPCR assay.
Thus, large-volume IMS based on the mPCR approach exhibits great potential
as a rapid, sensitive, and reliable diagnostic tool for the detection of common

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Identification