The revolution of agriculture through biotechnology have produced large-scale of genetically
modified crops which brought up a controversy on the safety usage of genetically modified
organisms (GMOs). It has been implemented globally that all GMO products and its derived
ingredients should have regulations on the usage and labelling. Thus, it is necessary to develop
methods that allow rapid screening of GMO products to comply with the regulations. This
study employed a reliable and flexible multiplex polymerase chain reaction (PCR) method for
the rapid detection of transgenic elements in genetically modified soy and maize along with
the soybean LECTIN gene and maize ZEIN gene respectively. The selected four common
transgenic elements were 35S promoter (35S); Agrobacterium tumefaciens nopaline synthase
terminator (NOS); 5-enolypyruvylshikimate-3-phosphate synthase (epsps) gene; and Cry1Ab
delta-endotoxin (cry1Ab) gene. Optimization of the multiplex PCR methods were carried out
by using 1% Roundup ReadyTM Soybean (RRS) as the certified reference material for soybean
that produced fourplex PCR method detecting 35S promoter, NOS terminator, epsps gene and
soybean LECTIN gene and by using 1% MON810 as the certified reference material for maize
that produced triplex PCR method detecting 35S promoter, cry1Ab gene and maize ZEIN gene
prior to screening of the GMO traits in various food products and animal feeds. 1/9 (11.1%) of
the animal feed contained maize and 1/15 (6.7%) of the soybean food products showed positive
results for the detection of GMO transgenic gene. None of the maize food products showed
positive results for GMO transgenic gene. In total, approximately 4% of the food products
and animal feed were positive as GMO. This indicated GMOs have not widely entered the
food chain. However, it is necessary to have an appropriate screening method due to GMOs’
unknown potential risk to humans and to animals. This rapid screening method will provide
leverage in terms of being economically wise, time saving and reliable.
In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.
Foodborne diseases are mainly caused by bacterial contamination which can lead to severe diarrhea. This study aimed to detect the presence of Shiga toxin-Producing Escherichia coli O157, Escherichia coli non-O157 and virulence gene in raw vegetables. The samples were purchased from wet market and hypermarket in Selangor. The detections were carried out by using the combination methods of Most Probable Number-Polymerase Chain Reaction (MPNPCR). A total of 37(18.5%) samples were found to be contaminated by STEC. Out of these 37 isolates, four (10.8%) of the isolates were E. coli O157 while 33(89.2%) were E. coli nonO157. However, there was no E. coli O157:H7 detected in all the samples. The occurrence of Shiga toxin-Producing E. coli in edible raw vegetables samples suggests the importance of this pathogen in vegetables. Therefore, more studies are required to remove this pathogen from vegetables.
The aim of this study was to assess the most probable number-polymerase chain reaction (MPNPCR) technique for detection of Listeria monocytogenes in salad vegetables in comparison with reference EN ISO 11290-2 and Food Drug Administration Bacteriological Analytical Manual method using artificial and naturally contaminated samples. Based on recovery of L. monocytogenes from artificially contaminated samples, MPN-PCR showed a moderate correlation (R=0.67) between spiking concentration and microbial levels which was better than the FDA-BAM method (R=0.642) and ISO 11290-2:1998 method (R=0.655). With naturally contaminated samples, it was found that L. monocytogenes was detected in 25% of the vegetable samples using MPN-PCR; 15% of the samples by the FDA-BAM method and 8% of samples using ISO 11290-2:1998 method. Overall, MPN-PCR was found to be a rapid and reliable method that could facilitate the enumeration of L. monocytogenes in vegetables.
The organic foods’ market is becoming one of the rapidly growing sections in agricultural economies in the world. During the last two decades, food-borne outbreaks associated with fresh produce have rapidly increased. E. coli O57:H7, the caustic agent of acute hemorrhagic diarrhea and abdominal cramps, is mainly associated with meat and poultry product outbreaks but frequent outbreaks linked to the consumption of vegetables have been reported. The aim of this study was to investigate prevalence of E. coli O157:H7 in some organic foods. A total of 230 organic food samples including four-winged bean, tomato, white radish, red cabbage, chinese cabbage, lettuce, cucumber and chicken form retailed groceries and supermarkets in Malaysia were investigated. Low prevalence of E. coli O157:H7 was detected in organic vegetables and chickens. The estimated quantity of E. coli O157:H7 in all samples ranged from 2400 MPN/g. The overall MPN/g estimate of E. coli O157:H7 in the samples from organic groceries was higher than supermarket with the maximum of >2400 MPN/g. Most of the samples from supermarket showed a minimum of
Salmonella has been reported to be presence both in raw and processed foods worldwide. In this study, the prevalence, quantification and antibiotic susceptibility of Salmonella isolated from raw vegetables or locally known as ulam such as asiatic pennywort (Centella asiatica (L) Urb), water dropwort (Oenanthe javanica (Blume) DC), long bean (Vigna sinensis EndL), and winged bean (Psophocarpus tetragonolobus (L) DC) obtained from retail markets in Selangor, Malaysia were carried out. From 96 samples tested, the overall prevalence of Salmonella spp. was 97.9%, Salmonella Enteritidis was 54.2% and Salmonella Typhimurium was 82.3% respectively. Samples were contaminated with Salmonella ranging from < 3 to 2400 MPN/g. Salmonella Enteritidis and Salmonella Typhimurium isolates obtained from the raw vegetables (ulam) were found to exhibit high resistance against ampicillin (100%), erythromycin (100%), amoxicillin/clavunic acid (81.3%), cephalothin (75%), streptomycin (50%) and ciprofloxacin (50%). All Salmonella isolates showed multi drug resistant (MDR) profile with each isolate being resistant to 3 or more antibiotics. The multiple antibiotic resistance (MAR) index of Salmonella isolates ranged from 0.27 to 0.55 for Salmonella Enteritidis and 0.27 to 0.82 for Salmonella Typhimurium. The presence of Salmonella on raw vegetables (ulam) and high antibiotic resistance isolates indicated that raw vegetables could be contaminated and thus imposes possible health risk to local consumers.
The prevalence of Salmonella in chicken and beef sold in retails outlets in Malaysia was
determined by analysing 312 raw beef and chicken meat samples including their processed
products. Samples purchased from supermarkets, butcher shops and wet market, which being
classified into raw, minced and processed chicken and beef. A total of 86 (27.6%) samples were
found positive for Salmonella spp., with chicken meat samples (40.4%) showed greater presence
compared to beef (15.4%). Highest presence of Salmonella were detected from wet market
samples (35.4%), followed by supermarket (26.9%) and butcher shop (21.3%). The prevalence
of Salmonella were higher in unpacked chicken meat (84.8%), followed by unpacked beef
(27.8%). Salmonella serovars were identified as S. Enteritidis, S. Hadar, S. Dublin, S. Anatum,
S. Stanley, S. Gallinarum, S. Choleraesuis and S. Typhimurium. Detection of 8 Salmonella
serovars showed possibilities of cross contamination in various sources either at slaughtering
house, processing plant or until storage at retails level. Improper cooking method on meats and
hygiene practices prior to consume should be avoided in order to ensure food safety before
ingestion.
A total of 112 burger patties (35 beef burger patties, 39 chicken burger patties and 38 fish burger patties) which are commercially available at retail level were investigated for the presence and number of Listeria monocytogenes. These samples were analyzed using MPN-PCR method and conventional culturing methods. L. monocytogenes was detected in 33.3% of chicken burger patties, 22.9% of beef patties, and 10.5% of fish patty samples. From all contaminated raw burger patties, the estimated count of L. monocytogenes was ranged from 3 to 75 MPN/g. The results suggest that burger act as a potential source of listeriosis if the contaminated burger patty is consumed without adequate cooking. The risk associated with consumption of these samples was found to be high particularly for processed food at retail level in Malaysia. Therefore, food manufacturers play an important role in monitoring the manufacturing process and conduct a periodical surveillance on microbiological quality assessment on the processing plants. Besides, there is a need to increase awareness of consumers and food handlers to practice proper cooking of the burger patties before the point of consumption, to reduce the risk of listeria infection.
The prevalence of Campylobacter spp. in retailed sushi were examined using the techniques of polymerase chain reaction (PCR) in combination with most probable number (MPN) to quantify the bacteria in 150 samples obtained from three supermarkets. The average prevalence of Campylobacter spp. in retailed sushi was 26.6% with 32%, 16% and 32% from supermarket I, II and III, respectively. Campylobacter jejuni was found to be the predominant species in retailed sushi with 82.49% of all Campylobacter spp. positive samples. Campylobacter coli was not detected in all samples. The maximum MPN number of Campylobacter spp. in retailed sushi purchased from supermarket I, II and III ranged from 3.6-11.0 MPN/g, 9.4->1100 MPN/g and 27-1100 MPN/g, respectively. The isolation of C. jejuni from a variety of ready-to-eat retail sushi may indicate that these products can act as possible vehicles for the dissemination of food-borne campylobacteriosis.
Little is known on the biosafety level of Vibrio spp. in freshwater fish in Malaysia. The purpose of this study was to investigate the prevalence and concentration of Vibrio spp. and V. parahaemolyticus in
freshwater fish using the Most Probable Number-Polymerase Chain Reaction (MPN-PCR) method. The study was conducted on 150 samples from two types of freshwater fish commonly sold at hypermarkets, i.e. Pangasius hypophthalmus (catfish) and Oreochromis sp. (red tilapia). Sampling was done on the flesh, intestinal tract and gills of each fish. The prevalence of Vibrio spp. and V. parahaemolyticus was found to be 98.67% and 24% respectively with higher percentages detected in samples from the gills followed by the intestinal tract and flesh. Vibrio spp. was detected in almost all red tilapia and catfish samples. V. parahaemolyticus was detected in 25% of the catfish samples compared to 22.6% of red tilapia fish. The density of Vibrio spp. and V. parahaemolyticus in the samples ranged from 0 to 1.1x107 MPN/g. Although the maximum value was 1.1x107 MPN/g, most samples had microbial loads ranging from 0 to >104 MPN/g. The outcome on the biosafety assessment of Vibrio spp. and V. parahaemolyticus in freshwater fish indicates another potential source of food safety issues to consumers.
The purpose of this study was to investigate the biosafety of Vibrio parahaemolyticus in raw salad vegetables at wet market and supermarket in Malaysia. A combination of Most Probable Number - Polymerase Chain Reaction (MPN-PCR) method was applied to detect the presence of V. parahaemolyticus and to enumerate their density in the food samples. The study analyzed 276 samples of common vegetables eaten raw in Malaysia (Wild cosmos = 8; Japanese parsley = 21; Cabbage = 30; Lettuce = 16; Indian pennywort = 17; Carrot = 31; Sweet potato = 29; Tomato = 38; Cucumber = 28; Four winged bean = 26; Long bean = 32). The samples were purchased from two supermarkets (A and B) and two wet markets (C and D). The occurrence of V. parahaemolyticus detected was 20.65%, with higher frequency of V. parahaemolyticus in vegetables obtained from wet markets (Wet market C = 27.27%Wet Market D = 32.05%) compared to supermarkets (Supermarket A = 1.64%; Supermarket B = 16.67%). V. parahaemolyticus was most prevalent in Indian pennywort (41.18%). The density of V. parahaemolyticus in all the samples ranged from <3 up to >2400 MPN/g, mostly <3 MPN/g concentration. Raw vegetables from wet markets contained higher levels of V. parahaemolyticus compared to supermarkets. V. parahaemolyticus were present in raw vegetables although in low numbers. The results suggest that raw vegetables act as a transmission route for V. parahaemolyticus. This study will be the first biosafety assessment of V. parahaemolyticus in raw vegetables in Malaysia.
The continued and increasing development of antimicrobial resistant bacteria among the
foodborne pathogens had caused worldwide to be alarmed. Being the earliest to develop
antimicrobial resistance, Staphylococcus aureus is constantly monitored for any new
resistance development. The resistance development is often linked to wastewater and the
treatment plants where the pressure of antibiotic is the highest. Hence, this study
investigated on the prevalence of high antimicrobial resistant S. aureus in the wastewater
eluted from a poultry slaughterhouse. A total of thirty wastewater samples were collected
from a poultry slaughterhouse in Semenyih, Selangor. Most probable number (MPN)-
plating method was employed to enumerate the S. aureus count in the wastewater. The
results indicated that S. aureus was highly present whereby all samples (100%) were
positive and the concentration ranged between 11 – 2.1 x 104 MPN/ml. Isolated S. aureus
strains were screened for their antimicrobial susceptibility using the Kirby-Bauer Disk
Diffusion Test method to classify their antimicrobial resistance eleven antibiotics. The
MAR index measured was between 0.18 and 0.91, inferring that the strains are highly
antimicrobial resistance. All S. aureus strains were 100% resistant to ampicillin (25 µg)
and cefazolin (30 µg). 94.1% of the strains were resistant to penicillin (10 µg) which
phenotypically indicated these strains are Methicillin-resistant S. aureus (MRSA).
Notably, 17.6% of the strains developed resistance to vancomycin and was categorized as
Vancomycin-resistant S. aureus (VRSA). There is a need to take drastic preventive
measures to control the resistance development in S. aureus to conserve public health.
Salmonella remains to be a major foodborne pathogen for animals and humans and is the
leading cause of foodborne infections and outbreaks in various countries. Salmonella Enteritidis
is one of the most frequently isolated serotypes in poultry and poultry products from human
food poisoning cases. It can cause mild to acute gastroenterititis as well as other common
food poisoning symptoms when infection takes place in human. Nucleic acid amplification
technologies such as Polymerase Chain Reaction (PCR) is a tool that is rapid and sensitive
for detection of bacterial pathogen. We report the successful detection of S. Enteritidis by
PCR in raw chicken meat artificially-contaminated with serial concentration of S. Enteritidis
using crude DNA extracts as DNA template. PCR primers, ENT-F and ENT-R targeted on sdfI
gene were used to amplify DNA region unique to S. Enteritidis with crude DNA extract of the
samples, yielded product with the size of 303 bp. These primers were specific to S. Enteritidis
when tested by in-silico simulation against genome database of targeted bacterial species and
confirmed in PCR as amplification bands were observed with S. Typhimurium, S. Polarum and
S. Gallinarum. The established PCR can detect as few as 9.4 X 101
CFU/ml of inoculated S.
Enteritidis concentration and proved that pre-enrichment effect have significant effect on PCR
detection by increasing 1000-fold of the sensitivity limit compared to the non pre-enriched
samples. The PCR technique indicated that it can be successfully coupled with pre-enrichment
step to offer advantage in routine screening and surveillance of bacterial contamination in food
samples.
Peanuts are widely consumed as the main ingredient in many local dishes in Malaysia. However, the tropical climate in Malaysia (high temperature and humidity) favours the growth of fungi from Aspergillus section Flavi, especially during storage. Most of the species from this section, such as A. flavus, A. parasiticus and A. nomius, are natural producers of aflatoxins. Precise identification of local isolates and information regarding their ability to produce aflatoxins are very important to evaluate the safety of food marketed in Malaysia. Therefore, this study aimed to identify and characterize the aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi in peanuts and peanut-based products. A polyphasic approach, consisting of morphological and chemical characterizations was applied to 128 isolates originating from raw peanuts and peanut-based products. On the basis of morphological characters, 127 positively identified as Aspergillus flavus, and the other as A. nomius. Chemical characterization revealed six chemotype profiles which indicates diversity of toxigenic potential. About 58.6%, 68.5%, and 100% of the isolates are positive for aflatoxins, cyclopiazonic acid and aspergillic acid productions respectively. The majority of the isolates originating from raw peanut samples (64.8%) were aflatoxigenic, while those from peanut-based products were less toxigenic (39.1%). The precise identification of these species may help in developing control strategies for aflatoxigenic fungi and aflatoxin contamination in peanuts, especially during storage. These findings also highlight the possibility of the co-occurrence of other toxins, which could increase the potential toxic effects of peanuts.
Local wood charcoal was used as the main component of the electrodes of an air-cathode microbial
fuel cell (air-cathode MFC) in current study. The air cathode was build with finely milled charcoal powder and cement plaster as binder; while anode was made up of a packed bed of charcoal granules. Mangrove estuary brackish water was inoculated in the anodic chamber as the fuel and a source of exoelectrogens. The constructed fuel cell was monitored by measuring the potential over time. The MFC generated a stable power density at 33mW/m2 (0.5V) under a load of 200Ω after 72 hours of operation. An open circuit voltage (OCV) of 0.7mV was obtained after 15 hours operating under open circuit. The result of power generation by the constructed fuel cell indicating that wood charcoal could be used as electrode in an MFC and that brackish water contained potential exoelectrogens. However, further investigation and modification is required to increase the performance of the fuel cell.
The objectives highlighted in the present study were to determine the estimates of measurement uncertainty associated with PALCAM and CHROMagarTM Listeria media, to compare the efficacy between both media in relation to their measurement uncertainties. In addition, this study was carried out to assess the performance characteristics of spread and spiral plating procedures based on the comparison of Listeria monocytogenes enumeration between PALCAM and CHROMagarTM Listeria media. This work involved pure culture experiment, artificially contaminated samples experiment and naturally contaminated samples experiment. In pure culture experiment, PALCAM performance was relatively inferior to CHROMagarTM Listeria medium for both plating procedures. From the artificially contaminated samples, the results revealed that the values of repeatability, reproducibility, and measurement uncertainty at 95% confidence interval were comparable between both media under evaluation. However, at the level of naturally contaminated samples, the performance of CHROMagar
TM Listeria medium was refutable as the presence of high number of competitive microorganisms reduced the clarity of the medium. The current emphasis in ensuring microbiological safety which requires use of accredited laboratories has led to measurable need for measurement uncertainty to ensure reliability of test results for global acceptance.
The main source of E. coli 0157:H7 is cattle, but recent studies showed high percentage of outbreaks
contributed by contaminated water. The occurrence of E. coli O157:H7 in environmental water samples poses a potential threat to human health. The aim of this study was to establish a protocol for the detection of the pathogen E. coli O157:H7 and E. coli virulence genes (eaeA, rfbE, hly, stx1, and stx2) in a multiplex PCR protocol using six specific primer pairs. The target genes produced species-specific amplicons at 625 bp, 397 bp, 296 bp, 166 bp, 210 bp and 484 bp for E. coli O157:H7 (fliCh7 gene) and virulence genes (eaeA, rfbE, hly, stx1, and stx2) respectively. The results obtained show that the established PCR protocol is suitable for a rapid and specific analysis of the pathogenic E. coli O157:H7 in environmental water samples for the assessment of microbiological risks.
Several Norovirus cases due to consumption of green onions have been reported during recent years but reports on red onions are not found. Onions are one of the major tastes in Malaysian food which are sometimes consuming raw especially the green onion. Viral contamination in onions can occur due to planting condition and not properly prepared food. This situation can pose the human health risk. A method was developed to detect the Norovirus that might present on different type of onions. In this study, 60 samples were collected from local market. Elution by Tryptose Phosphate Glycine broth and concentration steps using negatively charge filter were applied to enhance the detection of virus in food due to low copies of virus on food surface. The viral RNA was extracted using Qiagen Rneasy Mini kit before further detection using One-step RT-PCR. The total incidence of Norovirus in green onion and red onion was 13.33% (4/30) and 3.33 % (1/30) respectively. This is the first report of the detection of Norovirus in red and green onions in Malaysia. Based on the results, it is concluded that this method is reliable to detect Norovirus on onions and vegetables surface and hence can be applied in the laboratories for routine or food borne outbreak investigation.
Irrespective of its health effects, street foods are very popular with the consumers. The main
purpose of this research was to study the biosafety of Escherichia coli in popiah, a Malaysian
street food sold at a roadside food stall and a restaurant in Sri Serdang, Selangor, Malaysia,
using the combination of the most probable number (MPN)-Polymerase Chain Reaction
(PCR) assay-plating on Eosin Methylene Blue (EMB) agar methods. Using these biomolecular
methods, E. coli was detected in 12/15 (80%) and 11/15 (73%) of the collected samples from
the roadside food stall and the restaurant respectively. The incidence of stx virulence-associated
genes was detected in 1/15 (7%) among the E. coli isolated from samples taken from the
roadside food stall while the E. coli isolated from the restaurant was 3/15 (20%). The density
of E. coli ranged from 1100 MPN/g and the density of E. coli positive with stx genes
was
Broiler part samples (80 fresh and 80 chilled) were examined for the prevalence and numbers of C. jejuni and C. coli by employing most-probable-number (MPN) and polymerase chain reaction (PCR) techniques. The prevalence of the bacteria was high where C. jejuni was detected in 92.5% fresh and 53.8% chilled samples while C. coli in 80.0% fresh and 56.3% chilled. The number of these bacteria in the positive fresh and chilled samples was from 3 to more than 2400 MPN/g and from 3 to 290 MPN/g, respectively. Antibiotic resistance test (using Kirby-Bauer disc diffusion method) on 10 C. jejuni and 13 C. coli isolates toward ampicillin, tobramycin, enrofloxacin, ciprofloxacin, tetracycline, cephalothin, gentamicin and norfloxacin revealed high resistance toward all antibiotics (20.0% - 100.0%). All isolates were resistant to at least two antibiotics. This study highlights the potential of multidrug-resistant C. jejuni and C. coli transmission to humans through fresh and chilled broiler parts. Consecutive studies with bigger sample sizes and covering all over Malaysia are warranted in future.