Rapid Salmonella Detection in

Different Food
Samples by Direct-PCR

Valdy azhari/ 155100500111011


Introduction
The background of the researcher
doing the research and the
purpose of the research
Presentation Outline
Material and Method
Isolation and identification of the
salmonella from meat and chicken
product and direct PCR of the
product
Result and Discussion
The result of the research from
comparing direct pcr method and
determined the most rapid &
sensitive technique of detecting
salmonella, Discussion of the
research and the conclusion of the
research
Introduction
Research Background

Salmonella Species
Foodborne diseases are
a serious threat to public health

Salmonella species are considered to be among the most

important foodborne pathogens in the world and
salmonellosis is still one of the most widespread foodborne
bacterial illnesses in humans

How?
Research Background

Salmonella outbreaks are

linked to unhygienic food
preparation, cooking,
reheating and storage
practices

Salmonella contamination can be

detect by using Direct- PCR

More rapid, sensitive and also can identify strain

difference by targeting gene(s) or sequence
exhibiting variability in its distribution within the
bacterial population
Research Purpose

“a comparative study for rapid detection methods of Salmonella

in different food samples using Direct-PCR on food samples or
combined with pre-enrichment and / or selective enrichment
media with the standard conventional microbiological methods“
Material and Method
Isolation and Identification
placed in a sterile
stomacher bag containing
225 ml of Buffered
Peptone Water
Homogenized for 2 min at inoculated into tubes
320 rpm and overnight containing 10 ml Rappaport
incubation at 37°C, 0.1 ml Vassiliadis (RV) broth and
aliquots incubated for 48 h at 42 °C

25g each
Xylose Lysine Deoxycholate
sample (XLD) agar plates were
inoculated from each of the RV
Sample broths
A total of 400 samples were collected
as follows:
200 poultry byproducts (50 shawerma,
50 minced chicken meat, 50 chicken
Suspect colonies with
wings and legs and 50 chicken fillets) , typical Salmonella incubated for 18–
while 200 meat byproducts were 50 morphology were 24 h at 37°C
confirmed biochemically.
oriental sausage, 50 minced meat, 50
livers and kidneys and 50 frozen meat.
All samples were aseptically collected
and sent to the laboratory
Direct PCR
0.4 mm Tissue samples were Tube was mixed by briefly
place directly into the PCR
sample placed into 20 µL of vortexing and spun down the
reaction (50 µL of volume) solution
Dilution Buffer

Primer sequence of gene amplification Reaction was incubated for 2−5

minutes at room temperature
The gene that being amplificated was invA gene Salmonella and then placed into the
preheated (98°C) block for 2
contains sequences unique to this genus and has been minutes
proved as a suitable PCR target
invA gene of Salmonella 5´GTG AAA TTA TCG CCA CGT
TCG GGC AA -3´and 5´ TCA TCG CAC CGT CAA AGG AAC
C -3´ The remaining tissue was
spun down and 1 µL the
supernatant was sufficient
PCR Mix (Kit) PCR Condition for a 20 µL PCR reaction

each PCR amplification reaction was set in a
volume of 50 ml with 2.5 µL from the sample,
1 µL forward primer, 1 µL reverse primer, 12.5
µL Phire animal Tissue PCR buffer And
Distilled Water Up To 50 µL
Were as follows: 5 minute at 980 C for initial
denaturation, followed by 40 cycles of denaturation,
annealing and extension (98C/5 sec and 72C/20 sec),
followed by final extension for 1 min at 720 C.
Result and Discussion
Result
Result

• The less sensitive detection method was Direct-PCR on food samples 5% for
both chicken & meat samples, followed by conventional isolation method 7%
for chicken & 5% for meat. The most optimized technique was Direct-PCR on
selective & pre-enrichment together 9.5 & 11.5 % for chicken & meat samples,
respectively
Result
 Discussion

• The results show that greater numbers of samples positive for

Salmonella species were detected by the Direct-PCR on selective
broth (RV) by 9.5% & 11.5 for poultry & meat respectively
• followed by Direct-PCR on enrichment broth (BPW) by 8 for poultry
& 7 for meat and that the Direct-PCR on food samples was 5% for
poultry lower that standard methods 7%, while in meat the
detection percent was the same for Direct-PCR on food and
standard method (5%).
 CONCLUSION

• It was concluded that the Direct-PCR on 18 hours selective

proceeded by 24 hours buffered peptone water is more sensitive
and time decreasing approach necessary to detect and identify
Salmonella. The applied Direct-PCR on selective broth can be an
intense tool for the rapid and accurate detection and identification
of Salmonella and can be executed in diagnostic and food analysis
laboratories
Thank You!
ANY QUESTIONS?