Displaying publications 61 - 80 of 1868 in total

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  1. Fulltext A rapid and sensitive Loop-mediated isothermal amplification assay for detection of pork DNA based on porcine tRNA lys and ATPase 8 genes
    Abdullahi, U.F., Igwenagu, E., Aliyu, S., Mu’azu, A., Naim, R., Wan-Taib, W.R.
    International Food Research Journal, 2017;24(4):1357-1361.
    MyJurnal
    This study describes the development of a rapid and sensitive Loop-mediated isothermal
    amplification assay for detection of swine DNA in adulterated meat and meat products. The
    need to protect consumer’s right to eat foods of their choices, has made it imperative for
    researchers to develop efficient means of screening and certification of food products. Six sets
    of LAMP primers designed based on porcine tRNA lysine gene and ATPase subunit 8 genes
    were used for the assay. Amplification was carried out under constant temperature (630C), using
    a simple laboratory water bath. Average time spent in amplification and detection of results was
    25 min. All results were visually detected and confirmed by electrophoresis. Detection limit of
    the assay was 0.03 femtogram (fg) much high than the PCR assay, and detection probability of
    the assay was 100%. Detection of 0.5% of pork spiked with 99.5% of cattle beef is indicative
    of the sensitivity and robustness of the assay. This could serve as a prototype for development
    of a sensitive and inexpensive Swine DNA LAMP detection kit.
    Matched MeSH terms: Polymerase Chain Reaction
  2. Fulltext A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides
    A Talip B, Snelling WJ, Sleator RD, Lowery C, Dooley JSG
    BMC Microbiol, 2018 11 26;18(1):196.
    PMID: 30477427 DOI: 10.1186/s12866-018-1335-0
    BACKGROUND: The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target.

    RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM.

    CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  3. Fulltext A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages
    Teoh BT, Chin KL, Samsudin NI, Loong SK, Sam SS, Tan KK, et al.
    BMC Infect Dis, 2020 Dec 11;20(1):947.
    PMID: 33308203 DOI: 10.1186/s12879-020-05585-4
    BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.

    METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.

    RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P 

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  4. A review of CRISPR-Cas and PCR-based methods for the detection of animal species in the food chain-current challenges and future prospects
    Sultana S, Azlan A, Mohd Desa MN, Mahyudin NA, Anburaj A
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2024 Mar;41(3):213-227.
    PMID: 38284970 DOI: 10.1080/19440049.2024.2304577
    Regular testing and systematic investigation play a vital role to ensure product safety. Until now, the existing food authentication techniques have been based on proteins, lipids, and nucleic acid-based assays. Among various deoxyribonucleic acid (DNA)-based methods, the recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based bio-sensing is an innovative and fast-expanding technology. The CRISPR/Cas-9 is known as Clustered Regularly Interspaced Short Palindromic Repeats due to the flexibility and simplicity of the CRISPR/Cas9 site-specific editing tool has been applied in many biological research areas such as Gene therapy, cell line development, discovering mechanisms of disease, and drug discovery. Nowadays, the CRISPR-Cas system has also been introduced into food authentication via detecting DNA barcodes of poultry and livestock both in processed and unprocessed food samples. This review documents various DNA based approaches, in an accessible format. Future CRISPR technologies are forecast while challenges are outlined.
    Matched MeSH terms: Polymerase Chain Reaction
  5. A review on current diagnostic tools and potential optical absorption spectroscopy for HFMD detection
    Mustafa FH, Ismail I, Ahmad Munawar AAZ, Abdul Basir B, Shueb RH, Irekeola AA, et al.
    Anal Biochem, 2023 Dec 15;683:115368.
    PMID: 37890549 DOI: 10.1016/j.ab.2023.115368
    Hand, Foot, and Mouth Disease (HFMD) is an outbreak infectious disease that can easily spread among children under the age of five. The most common causative agents of HFMD are enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but infection caused by EV71 is more associated with fatalities due to severe neurological disorders. The present diagnosis methods rely on physical examinations by the doctors and further confirmation by laboratories detection methods such as viral culture and polymerase chain reaction. Clinical signs of HFMD infection and other childhood diseases such as chicken pox, and allergies are similar, yet the genetics and pathogenicity of the viruses are substantially different. Thus, there is an urgent need for an early screening of HFMD using an inexpensive and user-friendly device that can directly detect the causative agents of the disease. This paper reviews current HFMD diagnostic methods based on various target types, such as nucleic acid, protein, and whole virus. This was followed by a thorough discussion on the emerging sensing technologies for HFMD detection, including surface plasmon resonance, electrochemical sensor, and surface enhanced Raman spectroscopy. Lastly, optical absorption spectroscopic method was critically discussed and proposed as a promising technology for HFMD screening and detection.
    Matched MeSH terms: Polymerase Chain Reaction
  6. Fulltext A review on the global widespread of TTV infection among humans population
    Nur Syazwani Jarkasi, Zamberi Sekawi, Cheah, Yoke Kqueen, Zulkefley Othman
    The Pertanika Journal of Scholarly Research Reviews, 2018;4(1):10-24.
    MyJurnal
    Torque Teno Virus (TTV) is a human-infected virus that is present ubiquitously in nature. Globally, it infects up to 95% of the healthy individuals without any clinical manifestations. The widely used laboratory diagnosis of TTV infection is Polymerase chain reaction (PCR). Nevertheless, several other methods have been developed. The rapid growth of TTV variants over time has posed a challenge in estimating the global TTV infection as none of the PCR protocol has the ability to detect the entire spectrum of TTV variants. Multiple TTV epidemiological studies have been conducted among Asian population, whereas other continents showed a limited number of studies. The horizontal and vertical transmission of TTV among humans population, as well as interspecies transmission are potentially related to the global widespread of TTV infection.
    Matched MeSH terms: Polymerase Chain Reaction
  7. A simple 18S rDNA approach for the identification of cultured eukaryotic microalgae with an emphasis on primers
    Khaw YS, Khong NMH, Shaharuddin NA, Yusoff FM
    J Microbiol Methods, 2020 05;172:105890.
    PMID: 32179080 DOI: 10.1016/j.mimet.2020.105890
    Any forms of valorization of microorganisms would require accurate identity recognition to ensure repeatability, reproducibility and quality assurance. This study aimed to evaluate the effectiveness of different primers for identifying cultured eukaryotic microalgae using a simple 18S rDNA approach. A total of 34 isolated microalgae and one culture collection were utilized in the search for an effective molecular identification method for microalgae. Ammonium formate was applied to marine microalgae prior to DNA extraction. The microalgal DNA was extracted using a commercial kit and subjected directly to PCR amplification using four different published 18S rDNA primers. The DNA sequences were analysed using Basic Local Alignment Search Tool (BLAST) and phylogenetic trees to determine the microalgae identity. The identity was further validated with conventional morphological taxonomic identification, and the relationship of microalgal morphology and genetic materials was also determined. The microalgal DNA was successfully amplified, including marine species without prior cleaning. In addition, the ss5 + ss3 primer pair was found to be an ideal primer set among the tested primers for identifying microalgae. Overall, molecular identification showed relative matching with morphological identification (82.86%). This study is important because it serves as a platform to develop a standardized eukaryotic microalgae identification method. In addition, this method could help to ease the eukaryotic microalgae identification process and enrich the current reference databases such as GenBank.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  8. A simple PCR method for rapid genotype analysis of Mycobacterium ulcerans
    Stinear T, Davies JK, Jenkin GA, Portaels F, Ross BC, Oppedisano F, et al.
    J Clin Microbiol, 2000 Apr;38(4):1482-7.
    PMID: 10747130
    Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  9. Fulltext A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae
    Yee W, Abdul-Kadir R, Lee LM, Koh B, Lee YS, Chan HY
    3 Biotech, 2018 Aug;8(8):354.
    PMID: 30105179 DOI: 10.1007/s13205-018-1381-1
    In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.
    Matched MeSH terms: Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
  10. A simple and rapid genotyping method for beta-2 receptor (beta2 AR) gene using allele specific multiplex PCR
    Romaino SM, Teh LK, Zilfalil BA, Thong CP, Ismail AA, Amir J, et al.
    J Clin Pharm Ther, 2004 Feb;29(1):47-52.
    PMID: 14748897 DOI: 10.1046/j.1365-2710.2003.00535.x
    Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164).
    Matched MeSH terms: Polymerase Chain Reaction/methods
  11. A simple method for the detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase chain reaction
    Zainuddin Z, Teh LK, Suhaimi AW, Salleh MZ, Ismail R
    Clin Chim Acta, 2003 Oct;336(1-2):97-102.
    PMID: 14500040 DOI: 10.1016/s0009-8981(03)00319-x
    BACKGROUND: Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles.
    METHOD: Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results.
    RESULTS: We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence.
    CONCLUSION: This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  12. A simple multiplex PCR method for the concurrent detection of three CYP2C8 variants
    Muthiah YD, Lee WL, Teh LK, Ong CE, Salleh MZ, Ismail R
    Clin Chim Acta, 2004 Nov;349(1-2):191-8.
    PMID: 15469873 DOI: 10.1016/j.cccn.2004.06.024
    BACKGROUND: Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4.
    METHOD: Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers.
    RESULT: The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method.
    CONCLUSION: We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  13. Fulltext A simple protocol to isolate DNA from Malaysian stork blood collected on FTA® cards
    YEE, ELSIE Y. S., ZAINAL ZAHARI, AHMAD ISMAIL, YAP, C.K., TAN, S. G.
    Buletin Persatuan Genetik Malaysia, 2009;14(1):20-22.
    MyJurnal
    The blood of the Painted Storks (Mycteria leucocephala) and the Milky Storks (M. cinerea) from Malaysia were collected
    invasively from the breeding site. The blood was dropped on to FTA® cards and stored at room temperature. DNA was isolated from
    the FTA® cards through a modification of the Wizard DNA Purification kit (Promega) procedure and PCR was performed with 11 pairs
    of microsatellite primers of the American Wood Stork (M. americana). The collection of a drop of blood onto the card is superior to the
    usual practice of collecting about five ml of blood into a vacuum tube as it causes fewer traumas to these sensitive birds. Moreover, this
    collection procedure can be adopted for use in various wild animal species which are usually found in the remote areas of Malaysia as
    the sample collection cards can be transported back to the laboratory at room temperature. Our procedure allows the typing of several
    molecular genetic markers from just a drop of blood collected in the field and stored at room temperature alleviating the need for storage
    in expensive deep freezers or liquid nitrogen tanks.
    Matched MeSH terms: Polymerase Chain Reaction
  14. A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus
    Ghaznavi-Rad E, Nor Shamsudin M, Sekawi Z, van Belkum A, Neela V
    J Med Microbiol, 2010 Oct;59(Pt 10):1135-1139.
    PMID: 20616192 DOI: 10.1099/jmm.0.021956-0
    A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  15. A single, large deletion accounts for all the beta-globin gene mutations in twenty families from Sabah (North Borneo), Malaysia. Mutation in brief no. 240. Online
    Thong MK, Rudzki Z, Hall J, Tan JA, Chan LL, Yap SF
    Hum Mutat, 1999;13(5):413.
    PMID: 10338100 DOI: 10.1002/(SICI)1098-1004(1999)13:5<413::AID-HUMU15>
    Beta-thalassemia major is one of the commonest genetic disorders in South-East Asia. The spectrum of beta-thalassemia mutations in the various ethnic sub-populations on the island of Borneo is unknown. We studied 20 Dusun children from the East Malaysian state of Sabah (North Borneo) with a severe beta-thalassemia major phenotype, using a combination of Southern analysis, polymerase chain reaction analysis and direct sequencing. We found the children to be homozygous for a large deletion, which has a 5' breakpoint at position -4279 from the cap site of the beta-globin gene (HBB) with the 3' breakpoint located in a L1 family of repetitive sequences at an unknown distance from the beta-globin gene. This was similar to a recent finding of a large deletion causing beta-thalassemia first described in unrelated beta-thalassemia heterozygotes of Filipino descent. This report describes the first 20 families with homozygosity of the deletion causing a severe phenotype. It provides the first information on the molecular epidemiology of beta-thalassemia in Sabah. This finding has implications for the population genetics and preventative strategies for beta-thalassemia major for nearly 300 million individuals in South-East Asia.
    Matched MeSH terms: Polymerase Chain Reaction
  16. A single-gene approach for the subspecies classification of Mycobacteroides abscessus
    Ng HF, Ngeow YF
    Pathog Dis, 2020 11 11;78(8).
    PMID: 32945880 DOI: 10.1093/femspd/ftaa055
    The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.
    Matched MeSH terms: Polymerase Chain Reaction
  17. A study of association of the complement C4 mutations with systemic lupus erythematosus in the Malaysian population
    Puah SM, Lian LH, Chew CH, Chua KH, Tan SY
    Lupus, 2007;16(9):750-4.
    PMID: 17728371 DOI: 10.1177/0961203307079454
    The aim of the present study was to investigate the association of C4 gene mutations with systemic lupus erythematosus, in 130 Malaysian SLE patients and 130 healthy controls. Generally, various PCR approaches were used to screen the mutations of the C4 genes, which included 2 bp (+TC) insertions at codon 1213 in exon 29, 1 bp deletions (-C) at codon 811 in exon 20, 1 bp (-C), 2 bp (-GT) deletions at codons 522 and 497 in exon 13 and null alleles. No mutations located at exons 13, 20 and 29 of the C4 gene, were detected amongst the patient and control samples in this study. C4A*Q0 was found in two out of the 130 control samples, while C4B*Q0 was present in two out of the 130 SLE patients. Overall, our results do not demonstrate a significant association to these known C4 mutations identified by previous studies, in the Malaysian scenario.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  18. Fulltext A study on PCR-RFLP of Giardia duodenalis in Malaysia
    Latifah I, Teoh Ky, Wan KL, Normaznah Y, Rahmah M
    Malays J Pathol, 2007 Jun;29(1):25-31.
    PMID: 19105325 MyJurnal
    Giardia duodenalis causes diarrhoea and malabsorption. The objectives of the study were to detect local isolates of G. doudenalis by polymerase chain reaction (PCR) and to determine their restriction fragment length polymorphisms (RFLP). G. doudenalis isolated from stools of patients from Hospital Orang Asli Gombak were cultured axenically using TYI-S-33 medium with 10% foetal calf serum. The commercially designed primer-pair 432/433 was used to amplify a 0.52 kb segment known to encode the homologous cysteine-rich trophozoite surface antigen (tsp11 and tsa417). Results showed that the primer-pair 432/433 could amplify the target region of the local isolates. RFLP study on the identical isolates showed that all the restriction enzymes tested ( HindIII, ClaI, PstI and Kpn) gave a banding pattern similar to that of the WB strain a reference pathogenic strain from human. The reference pathogenic strain were commercially obtained from the American Type Culture Collection (ATCC).
    Matched MeSH terms: Polymerase Chain Reaction*
  19. A suitable method for the detection of a potential fraud of bringing macaque monkey meat into the food chain
    Rashid NR, Ali ME, Hamid SB, Rahman MM, Razzak MA, Asing, et al.
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(7):1013-22.
    PMID: 25906074 DOI: 10.1080/19440049.2015.1039073
    Being the third-largest primate population has not made macaque (Macaca fascicularis sp.) monkeys less exposed to threats and dangers. Despite wildlife protection, they have been widely hunted and consumed in several countries because of their purported nutritional values. In addition to trading as pure bush meats in several places, monkey meat has been sold in meatball and soup products in Indonesia. Thus the possibility of macaque meat trafficking under the label of common meats is quite high. This paper reports the development of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the shortest amplicon length for the confirmed detection of monkey meat under compromised states which are known to degrade DNA. We amplified a 120-bp region of d-loop gene using a pair of macaque-specific primers and confirmed their specificity for the target species through cross-challenging against 17 different species using a 141-bp site of an 18 S rRNA gene as an endogenous control for eukaryotes. This eliminated the possibilities of any false-negative detection with complex matrices or degraded specimens. The detection limit was 0.00001 ng DNA in a pure state and 0.1% of meat in mixed matrices and commercial meatball products. RFLP analysis further authenticated the originality of the PCR product and distinctive restriction patterns were found upon AluI and CViKI-1 digestion. A micro-fluidic lab-on-a-chip automated electrophoretic system separated the fragments with high resolution. The assay was validated for screening commercial meatball products with sufficient internal control.
    Matched MeSH terms: Polymerase Chain Reaction/methods; Polymerase Chain Reaction/veterinary*
  20. A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain
    Ali ME, Asing, Hamid SB, Razzak MA, Rashid NR, Al Amin M, et al.
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2015;32(8):1223-33.
    PMID: 26062948 DOI: 10.1080/19440049.2015.1058535
    Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005. However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.
    Matched MeSH terms: Polymerase Chain Reaction/veterinary
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