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Potential authentication of various meat-based products using simple and efficient DNA extraction method

Khairil Mokhtar NF, El Sheikha AF, Azmi NI, Mustafa S

J Sci Food Agric, 2020 Mar 15;100(4):1687-1693.
PMID: 31803942 DOI: 10.1002/jsfa.10183

BACKGROUND: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR.
RESULTS: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R2 = 0.9979) based on the regression analysis of the standard curve have been obtained.
CONCLUSION: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market. © 2019 Society of Chemical Industry.
Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules

Mohamad NA, Mustafa S, Khairil Mokhtar NF, El Sheikha AF

J Sci Food Agric, 2018 Sep;98(12):4570-4577.
PMID: 29505123 DOI: 10.1002/jsfa.8985

BACKGROUND: The pharmaceutical industry has boosted gelatin consumption worldwide. This is supported by the availability of cost-effective gelatin production from porcine by-products. However, cross-contamination of gelatin materials, where porcine gelatin was unintentionally included in the other animal sources of gelatin, has caused significant concerns about halal authenticity. The real-time polymerase chain reaction (PCR) has enabled a highly specific and sensitive animal species detection method in various food products. Hence, such a technique was employed in the present study to detect and quantify porcine DNA in gelatin using a molecular beacon probe, with differences in performance between mitochondrial (cytochrome b gene) and chromosomal DNA-(MPRE42 repetitive element) based porcine-specific PCR assays being compared.
RESULTS: A higher sensitivity was observed in chromosomal DNA (MPRE-PCR assay), where this assay allows the detection of gelatin DNA at amounts as as low as 1 pg, whereas mitochondrial DNA (CBH-PCR assay) can only detect at levels down to 10 pg of gelatin DNA. When an analysis with commercial gelatin and gelatin capsule samples was conducted, the same result was observed, with a significantly more sensitive detection being provided by the repetitive element of chromosomal DNA.
CONCLUSION: The present study has established highly sensitive DNA-based porcine detection systems derived from chromosomal DNA that are feasible for highly processed products such as gelatin and gelatin capsules containing a minute amount of DNA. This sensitive detection method can also be implemented to assist the halal authentication process of various food products available on the market. © 2018 Society of Chemical Industry.
Modification of gelatin-DNA interaction for optimised DNA extraction from gelatin and gelatin capsule

Mohamad NA, Mustafa S, El Sheikha AF, Khairil Mokhtar NF, Ismail A, Ali ME

J Sci Food Agric, 2016 May;96(7):2344-51.
PMID: 26441285 DOI: 10.1002/jsfa.7482

Poor quality and quantity of DNA extracted from gelatin and gelatin capsules often causes failure in the determination of animal species using PCR. Gelatin, which is mainly derived from porcine and bovine, has been a matter of concern among customers in order to fulfill religious obligation and safety precaution against several transmissible infectious diseases associated with bovine species. Thus, optimised DNA extraction from gelatin is very important for successful real-time PCR detection of gelatin species. In this work, the DNA extraction method was optimised in terms of lysis incubation period and inclusion of pre-treatment pH modification of samples.
Detection of porcine pepsin in model cheese using polyclonal antibody-based ELISA

Raja Nhari RMH, Muhammad Zailani AN, Khairil Mokhtar NF, Hanish I

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2020 Apr;37(4):561-567.
PMID: 32027553 DOI: 10.1080/19440049.2020.1717645

The usage of porcine pepsin or other porcine derivatives in food products is a common practice in European, American and certain Asian countries although it creates issues in religious and personnel health concerns. In this study, porcine pepsin was detected using indirect ELISA that involved the anti-pep80510 polyclonal antibody raised against a specific peptide of porcine pepsin, pep80510. The sensitivity of the assay for standard porcine pepsin was 0.008 µg/g. The immunoassay did not cross-react to other animal rennet and milk proteins except for microbial coagulant from Mucor miehie. The recovery of porcine pepsin in spiked cheese curd within the range of CV < 20% while for porcine pepsin in spiked cheese whey the recovery is also within the range of CV% < 20%.
Monoclonal antibody-based enzyme immunoassay for detection of porcine plasma in fish surimi

Raja Nhari RMH, Khairil Mokhtar NF, Hanish I, Hamid M, Mohamed Rashidi MAA, Shahidan NM

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 May;35(5):807-817.
PMID: 29285986 DOI: 10.1080/19440049.2017.1420920

Detection of porcine plasma using indirect ELISA was developed using mAb B4E1 for the prevention of their usage in human food that creates religious and health conflicts. The immunoassay has a CV
Halal authentication using lateral flow devices for detection of pork adulteration in meat products: a review

Raja Nhari RMH, Soh JH, Khairil Mokhtar NF, Mohammad NA, Mohd Hashim A

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2023 Aug;40(8):971-980.
PMID: 37535014 DOI: 10.1080/19440049.2023.2242955

Lateral flow devices (LFDs) are straightforward scientific tools that have made substantial advances in recent years. They have been used in many fields including the meat industry to detect disease markers, determine meat freshness or meat species determination. They are, therefore, significant in the research of meat adulteration by mixed animal species, because food component authenticity is a serious concern encompassing health, economic, legal, and religious issues. Pork adulteration is one of the most crucial issues in the global meat industry. In this review, we discuss the various types of LFDs and recent research on the development of LFDs as an authenticity tool for detecting pig additives in meat-based products, and how regulatory authorities could adopt LFDs for their workflows. Despite the benefits of rapidity, simplicity, low cost, high sensitivity, and specificity, researchers face challenges when using LFD as a final confirmation test. Future directions are suggested for globalising the use of LFD as a halal authentication method.
Rapid porcine detection in gelatin-based highly processed products using loop mediated isothermal amplification

Tasrip NA, Mohd Desa MN, Khairil Mokhtar NF, Sajali N, Mohd Hashim A, Ali ME, et al.

J Food Sci Technol, 2021 Dec;58(12):4504-4513.
PMID: 34629514 DOI: 10.1007/s13197-020-04932-2

Low DNA concentration recovered from highly processed products such as gelatin and gelatin-based products renders difficulty in detecting porcine contamination using conventional PCR techniques. We documented here a porcine-specific loop-mediated isothermal amplification (LAMP) to identify porcine traces in gelatin products. The porcine-specific primers were designed according to mitochondrial DNA of Cytochrome b gene sequence. Here we used two different reaction mixtures for LAMP assay (GENIE and MYRM) against the same DNA samples extracted from gelatin products and porcine-specific primers to detect the presence of porcine DNA. The porcine-specific primers were shown to be specific only to Sus scrofa against 14 DNA of other meat species. The analytical sensitivity of the LAMP assay for porcine DNA detection is 1 pg/µL using both GENIE (within 30 m) and MYRM (within 60 m) reaction mixtures. Analysis against 32 samples of gelatin products showed that five samples were found to contain porcine DNA; two samples out of six gelatin powder samples and three gelatin capsule samples out of nine. Out of these five positive samples, three were not labeled containing porcine gelatin. Overall, LAMP assay in this study showed an excellent specificity, sensitivity and rapidity in detection of porcine DNA in gelatin products.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-020-04932-2).
Effect of natural antibacterial clays against single biofilm formation by Staphylococcus aureus and Salmonella Typhimurium bacteria on a stainless-steel surface

Wan Omar WH, Mahyudin NA, Azmi NN, Mahmud Ab Rashid NK, Ismail R, Mohd Yusoff MHY, et al.

Int J Food Microbiol, 2023 Jun 02;394:110184.
PMID: 36996693 DOI: 10.1016/j.ijfoodmicro.2023.110184

Staphylococcus aureus and Salmonella Typhimurium have a propensity to develop biofilms on food contact surfaces, such as stainless-steel, that persist despite rigorous cleaning and sanitizing procedures. Since both bacterial species pose a significant public health risk within the food chain, improved anti-biofilm measures are needed. This study examined the potential of clays as antibacterial and anti-biofilm agents against these two pathogens on appropriate contact surfaces. Natural soil was processed to yield leachates and suspensions of both untreated and treated clays. Soil particle size, pH, cation-exchange capacity, and metal ions were characterized to assess their importance in bacterial killing. Initial antibacterial screening was performed on nine distinct types of natural Malaysian soil using a disk diffusion assay. Untreated leachate from Kuala Gula and Kuala Kangsar clays were found to inhibit S. aureus (7.75 ± 0.25 mm) and Salmonella Typhimurium (11.85 ± 1.63 mm), respectively. The treated Kuala Gula suspension (50.0 and 25.0 %) reduced S. aureus biofilms by 4.4 and 4.2 log at 24 and 6 h, respectively, while treated Kuala Kangsar suspension (12.5 %) by a 4.16 log reduction at 6 h. Although less effective, the treated Kuala Gula leachate (50.0 %) was effective in removing Salmonella Typhimurium biofilm with a decrease of >3 log in 24 h. In contrast to Kuala Kangsar clays, the treated Kuala Gula clays contained a much higher soluble metal content, especially Al (301.05 ± 0.45 ppm), Fe (691.83 ± 4.80 ppm) and Mg (88.44 ± 0.47 ppm). Elimination of S. aureus biofilms correlated with the presence of Fe, Cu, Pb, Ni, Mn and Zn irrespective of the pH of the leachate. Our findings demonstrate that a treated suspension is the most effective for eradication of S. aureus biofilms with a potential as a sanitizer-tolerant, natural antibacterial against biofilms for applications in the food industry.
Fulltext The characteristics and roles of antimicrobial peptides as potential treatment for antibiotic-resistant pathogens: a review

Zainal Baharin NH, Khairil Mokhtar NF, Mohd Desa MN, Gopalsamy B, Mohd Zaki NN, Yuswan MH, et al.

PeerJ, 2021;9:e12193.
PMID: 35003909 DOI: 10.7717/peerj.12193

The emergence of antibiotic-resistant bacteria has become a significant and ever-increasing threat to global public health, increasing both morbidity and mortality rates, and the financial burden on health services. Infection by drug-resistant bacteria is anticipated to contribute to the demise of almost 10 million people by the year 2050 unless a competent and effective response is devised to engage with this issue. The emergence and spread of resistance are commonly caused by the excessive or inappropriate use of antibiotics and substandard pharmaceuticals. It arises when pathogens adapt to different conditions and develop self-defence mechanisms. Currently, novel antimicrobial peptides (AMPs) have been reported to be the sole cure for some clinical cases of infectious diseases such as sepsis and skin infections, although these agents may, on occasion, require administration together with an adjunctive low-dose antibiotic. Although AMPs are a promising alternative form of anti-microbial therapy and easily applied in the medical sector, they still have limitations that should not be taken lightly. Hence, this review explores the characteristics, advantages and disadvantages of AMPs for their potential in treating antibiotic-resistant pathogens.