Displaying publications 41 - 60 of 1868 in total

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  1. Molecular detection of enteroviruses from an outbreak of hand, foot and mouth disease in Malaysia in 1997
    Abubakar S, Chee HY, Shafee N, Chua KB, Lam SK
    Scand. J. Infect. Dis., 1999;31(4):331-5.
    PMID: 10528868
    Enterovirus 5'UTR sequences were detected by RT-PCR in 22 out of 47 suspected hand, foot and mouth disease (HFMD) patients during an outbreak of the disease with incidences of fatal brainstem encephalomyelitis in Malaysia in 1997. Genetic and phylogenetic analyses of the isolates 5'UTR sequences suggest the presence of predominantly enteroviruses with high sequence similarities to Echovirus 1 and Coxsackievirus A9 in the Malaysian peninsula. No fatal cases, however, were associated with these isolates. The remaining isolates, including all (4/4) isolates of the fatal cases from the Malaysian peninsula and Sarawak shared very high sequence identity with enterovirus 71MS (EV71). These findings suggest that several enteroviruses were circulating in Malaysia during the outbreak period, with only EV71 causing fatal infections.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction*
  2. Molecular characterization and phylogeny of Shiga toxin-producing E. coli (STEC) from imported beef meat in Malaysia
    Abuelhassan NN, Mutalib SA, Gimba FI, Yusoff WM
    Environ Sci Pollut Res Int, 2016 Sep;23(17):17553-62.
    PMID: 27234829 DOI: 10.1007/s11356-016-6954-0
    This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak of food-borne disease. The isolation of pathogenic E. coli strains from the imported meat samples calls for prudent management of imported meats by the relevant authorities.
    Matched MeSH terms: Polymerase Chain Reaction
  3. Development of single-locus DNA microsatellite markers using 5'anchored ISSR-PCR method for the mangrove horseshoe crab, Carcinoscorpius rotundicauda (Latreille, 1802) in Peninsular Malaysia
    Adibah AB, Ling LP, Tan SG, Faridah QZ, Christianus A
    Mol Biol Rep, 2012 Apr;39(4):3815-20.
    PMID: 21744263 DOI: 10.1007/s11033-011-1159-6
    Horseshoe crabs are said to be declining worldwide. However, there is still no published report on the status of horseshoe crabs in Malaysia. Thus, we report here eight informative microsatellite markers that were developed using the 5'-anchored ISSR-PCR enrichment procedure to diagnose the population genetic structure of the mangrove horseshoe crab, Carcinoscorpius rotundicauda from Peninsular Malaysia. This set of markers was tested on 127 samples and showed polymorphism in this species. Hence they should be useful in future essential population genetic studies of these living fossils in the Southeast Asian region.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  4. Reversible thermo-pneumatic valves on centrifugal microfluidic platforms
    Aeinehvand MM, Ibrahim F, Harun SW, Kazemzadeh A, Rothan HA, Yusof R, et al.
    Lab Chip, 2015 Aug 21;15(16):3358-69.
    PMID: 26158597 DOI: 10.1039/c5lc00634a
    Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  5. First Report of Xanthomonas oryzae pv. oryzicola Causing Bacterial Leaf Streak of Rice in Burundi
    Afolabi O, Milan B, Amoussa R, Koebnik R, Poulin L, Szurek B, et al.
    Plant Dis, 2014 Oct;98(10):1426.
    PMID: 30703943 DOI: 10.1094/PDIS-05-14-0504-PDN
    On May 9, 2013, symptoms reminiscent of bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola were observed on rice plants at the panicle emergence stage at Musenyi, Gihanga, and Rugombo fields in Burundi. Affected leaves showed water-soaked translucent lesions and yellow-brown to black streaks, sometimes with visible exudates on leaf surfaces. Symptomatic leaves were ground in sterile water and the suspensions obtained were subjected to a multiplex PCR assay diagnostic for X. oryzae pathovars (3). Three DNA fragments (331, 691, and 945 bp) corresponding to X. oryzae pv. oryzicola were observed after agarose gel electrophoresis. Single bacterial colonies were then isolated from surface-sterilized, infected leaves after grinding in sterile water and plating of 10-fold dilutions of the cell suspension on semi-selective PSA medium (4). After incubation at 28°C for 5 days, each of four independent cultures yielded single yellow, mucoid Xanthomonas-like colonies (named Bur_1, Bur_2, Bur_6, and Bur_7) that resembled the positive control strain MAI10 (1). These strains originated from Musenyi (Bur_1), Gihanga (Bur_2), and Rugumbo (Bur_6 and Bur_7). Multiplex PCR assays on the four putative X. oryzae pv. oryzicola strains yielded the three diagnostic DNA fragments mentioned above. All strains were further analyzed by sequence analysis of portions of the gyrB gene using the universal primers gyrB1-F and gyrB1-R for PCR amplification (5). The 762-bp DNA fragment was identical to gyrB sequences from the Asian X. oryzae pv. oryzicola strains BLS256 (Philippines), ICMP 12013 (China), LMG 797 and NCPPB 2921 (both Malaysia), and from the African strain MAI3 (Mali) (2). The partial nucleotide sequence of the gyrB gene of Bur_1 was submitted to GenBank (Accession No. KJ801400). Pathogenicity tests were performed on greenhouse-grown 4-week-old rice plants of the cvs. Nipponbare, Azucena, IRBB 1, IRBB 2, IRBB 3, IRBB 7, FKR 14, PNA64F4-56, TCS 10, Gigante, and Adny 11. Bacterial cultures were grown overnight in PSA medium and re-suspended in sterile water (1 × 108 CFU/ml). Plants were inoculated with bacterial suspensions either by spraying or by leaf infiltration (1). For spray inoculation, four plants per accession and strain were used while three leaves per plant and four plants per accession and strain were inoculated by tissue infiltration. After 15 days of incubation in a BSL-3 containment facility (27 ± 1°C with a 12-h photoperiod), the spray-inoculated plants showed water-soaked lesions with yellow exudates identical to those seen in the field. For syringe-infiltrated leaves, the same symptoms were observed at the infiltrated leaf area. Re-isolation of bacteria from symptomatic leaves yielded colonies with the typical Xanthomonas morphology that were confirmed by multiplex PCR to be X. oryzae pv. oryzicola, thus fulfilling Koch's postulates. Bur_1 has been deposited in the Collection Française de Bactéries Phytopathogènes as strain CFBP 8170 ( http://www.angers-nantes.inra.fr/cfbp/ ). To our knowledge, this is the first report of X. oryzae pv. oryzicola causing bacterial leaf streak on rice in Burundi. Further surveys will help to assess its importance in the country. References: (1) C. Gonzalez et al., Mol. Plant Microbe Interact. 20:534, 2007. (2) A. Hajri et al. Mol. Plant Pathol. 13:288, 2012. (3) J. M. Lang et al. Plant Dis. 94:311, 2010. (4) L. Poulin et al. Plant Dis. 98:1423, 2014. (5) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction
  6. Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain
    Ahamad MNU, Ali ME, Hossain MAM, Asing A, Sultana S, Jahurul MHA
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2017 Dec;34(12):2043-2057.
    PMID: 28748739 DOI: 10.1080/19440049.2017.1359752
    Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction*
  7. Application of multilocus sequence analysis for molecular characterization of enterococci with virulence factors recovered from a tropical recreational beach
    Ahmad A, Dada AC, Usup G
    Southeast Asian J. Trop. Med. Public Health, 2014 May;45(3):700-18.
    PMID: 24974655
    Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.
    Matched MeSH terms: Polymerase Chain Reaction
  8. Fulltext Extraction of mitochondrial DNA from tooth dentin: application of two techniques
    Ahmad Azlina, Berahim Zurairah, Sidek Mohamad Ros, Mokhtar Khairani Idah, Samsudin Abdul Rani
    Archives of Orofacial Sciences, 2011;6(1):9-14.
    MyJurnal
    Mitochondrial DNA (mtDNA) is a hereditary material located in mitochondria and is normally maternally inherited. Mutational analysis performed on mtDNA proved that the mutations are closely related with a number of genetic illnesses, besides being exploitable for forensic identification. Those findings imply the importance of mtDNA in the scientific field. MtDNA can be found in abundance in tooth dentin where it is kept protected by the enamel, the hardest outer part of the tooth. In this study, two techniques of mtDNA extraction were compared to determine the efficacy between the two techniques. Teeth used for the study was collected from Dental Clinic, Hospital Universiti Sains Malaysia. After the removal of tooth from the tooth socket of the patient, the tooth was kept at -20C until use. Later, pulp tissue and enamel was excised using dental bur and only the root dentin was utilized for the isolation of mtDNA by crushing it mechanically into powdered form. MtDNA was extracted using the two published methods, Pfeifer and Budowle and then subjected to spectrophotometry DNA quantification and purity, Polymerase chain reaction (PCR) amplification of hypervariable-two region of mtDNA, followed by DNA sequencing to analyze the reliability of the extraction techniques. In conclusion, both techniques proved to be efficient and capable for the extraction of mtDNA from tooth dentin.
    Matched MeSH terms: Polymerase Chain Reaction
  9. CYP3A4*18 polymorphisms and anti-hepatitis A virus seroprevalence
    Ahmad F, Che Hamzah NA, Mustaffa N, Hua GS
    Hepatogastroenterology, 2011 07 15;58(110-111):1725-9.
    PMID: 21940338 DOI: 10.5754/hge11107
    BACKGROUND/AIMS: CYP3A4 is the major cytochrome in humans which shows reduced activity in chronic liver disease as well as in hepatic cirrhosis. The detection of this polymorphism may give an indication on the prognosis of patients having chronic viral hepatitis with superimposed hepatitis A infection. The aim of this study is to correlate the seroprevalence of anti-HAV antibodies in chronic liver disease patients having CYP3A4*18 polymorphisms.

    METHODOLOGY: This is a prospective study where patients (n=119) blood was tested for anti-HAVIgG and CYP3A4*18 polymorphism.

    RESULTS: The overall anti-HAV seroprevalence was 88.2%. The etiology of CLD was hepatitis B in 96 patients (80.7%) and hepatitis C in 23 patients (19.3%). There was a significant increase in the age of the prevalence of this disease after 30 years of age (p=0.008). CYP3A4*18 polymorphism was detected in 3 (2.5%) of the patients with chronic liver disease. However, there was no significant association between CP3A4*18 mutation and anti-HAV serology.

    CONCLUSIONS: Age was the most important factor in determining anti-HAV positivity. It is concluded that CYP3A4*18 genetic polymorphism does not play a main role in influencing the seroprevalence of anti-hepatitis A among chronic viral hepatitis B and C liver disease patients.

    Matched MeSH terms: Polymerase Chain Reaction
  10. Fulltext Diagnostic Potential of an IgE-ELISA in Detecting Strongyloidiasis
    Ahmad H, Balachandra D, Arifin N, Nolan TJ, Lok JB, Hayat Khan A, et al.
    Am J Trop Med Hyg, 2020 12;103(6):2288-2293.
    PMID: 32996454 DOI: 10.4269/ajtmh.20-0265
    Strongyloides stercoralis infection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate. However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial Strongyloides IgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate. Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11). Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n = 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative. The optical density values of group IB sera were also significantly lower than those of group IA (P < 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  11. Fulltext Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1)
    Ahmad MK, Tabana YM, Ahmed MA, Sandai DA, Mohamed R, Ismail IS, et al.
    Malays J Med Sci, 2017 Dec;24(6):29-38.
    PMID: 29379384 DOI: 10.21315/mjms2017.24.6.4
    Background: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication.

    Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.

    Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.

    Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

    Matched MeSH terms: Polymerase Chain Reaction
  12. Fulltext The discrimination of d-tartrate positive and d-tartrate negative S. enterica subsp. enterica serovar Paratyphi B isolated in Malaysia by phenotypic and genotypic methods
    Ahmad N, Hoon ST, Ghani MK, Tee KY
    Malays J Pathol, 2012 Jun;34(1):35-9.
    PMID: 22870596 MyJurnal
    Serotyping is not sufficient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identified all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  13. Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines
    Ahmad Nizar NN, Hossain M, Sultana S, Ahamad MN, Johan MR, Ali ME
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2019 Jun;36(6):825-835.
    PMID: 30945985 DOI: 10.1080/19440049.2019.1584407
    Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3-120.2% target recovery at 0.1-10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction*
  14. Double gene targeting PCR assay for the detection of Crocodylus porosus in commercial products
    Ahmad Nizar NN, Ali ME, Hossain MAM, Sultana S, Ahamad MNU
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Jun;35(6):1038-1051.
    PMID: 29447579 DOI: 10.1080/19440049.2018.1440644
    The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77-127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen.
    Matched MeSH terms: Polymerase Chain Reaction*
  15. Fulltext Detection of dengue virus from field Aedes aegypti and Aedes albopictus adults and larvae
    Ahmad R, Ismail A, Saat Z, Lim LH
    Southeast Asian J. Trop. Med. Public Health, 1997 Mar;28(1):138-42.
    PMID: 9322296
    Mosquito adults and larvae were collected from dengue high risk areas and transported to the laboratory for identification. Identified mosquitos were pooled according to the species, date and locality and stored at -70 degrees C. A total of 1,385 pools of Aedes albopictus and 267 pools of Ae. Aegypti were collected from major towns in 12 states in Peninsular Malaysia. Virus isolation was carried out using cell culture (C6/36 clone) of Ae. albopictus and detection of dengue virus by the peroxidase anti-peroxidase staining. All positive isolations were further re-confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Most of the pools were negative with PAP staining and RT-PCR. However, 11 mosquito pools were positive with PAP staining. On the other hand, samples from Terengganu, Pulau Pinang and Johor were positive using both methods.
    Matched MeSH terms: Polymerase Chain Reaction
  16. Plasmodium knowlesi - an emerging pathogen
    Ahmed MA, Cox-Singh J
    ISBT science series, 2015 Apr;10(Suppl 1):134-140.
    PMID: 26029250
    Ten years have passed since the publication of a large focus of Plasmodium knowlesi infections in the human population. The discovery was made during a molecular investigation of atypical P. malariae cases in the Kapit Health Division, Sarawak, Malaysian Borneo. Patients were more symptomatic with higher parasite counts than expected in P. malariae infections. The investigation found only P. knowlesi DNA present in patient blood samples. Morphological similarity had allowed P. knowlesi to masquerade as P. malariae during routine diagnostic microscopy for malaria. P. knowlesi, a malaria parasite of macaque monkeys, had entered the human population. The subsequent development of P. knowlesi species-specific PCR assays soon demonstrated that the entry was not confined to the Kapit Division but extended across island and mainland Southeast Asia. Relevant clinical descriptions and guidelines for the treatment and management of patents with P. knowlesi malaria were not available. Nor was it clear whether P. knowlesi had undergone a host switch event into the human population or if infections were zoonotic. The outputs of studies on P. knowlesi malaria during the past 10 years will be summarized, highlighting major findings within the context of pathophysiology, virulence, host switch events, treatment, control and importantly malaria elimination.
    Matched MeSH terms: Polymerase Chain Reaction
  17. Fulltext Utilization of Small RNA Genes to Distinguish Vibrio cholerae Biotypes via Multiplex Polymerase Chain Reaction
    Ahmed SA, Raabe CA, Cheah HL, Hoe CH, Rozhdestvensky TS, Tang TH
    Am J Trop Med Hyg, 2019 06;100(6):1328-1334.
    PMID: 30963989 DOI: 10.4269/ajtmh.18-0525
    The diarrheal disease "cholera" is caused by Vibrio cholerae, and is primarily confined to endemic regions, mostly in Africa and Asia. It is punctuated by outbreaks and creates severe challenges to public health. The disease-causing strains are most-often members of serogroups O1 and O139. PCR-based methods allow rapid diagnosis of these pathogens, including the identification of their biotypes. However, this necessitates the selection of specific target sequences to differentiate even the closely related biotypes of V. cholerae. Oligonucleotides for selective amplification of small RNA (sRNA) genes that are specific to these V. cholerae subtypes were designed. The resulting multiplex PCR assay was validated using V. cholerae cultures (i.e., 19 V. cholerae and 22 non-V. cholerae isolates) and spiked stool samples. The validation using V. cholerae cultures and spiked stool suspensions revealed detection limits of 10-100 pg DNA per reaction and 1.5 cells/mL suspension, respectively. The multiplex PCR assay that targets sRNA genes for amplification enables the sensitive and specific detection, as well as the differentiation of V. cholerae-O1 classical, O1 El Tor, and O139 biotypes. Most importantly, the assay enables fast and cheaper diagnosis compared with classic culture-based methods.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction*
  18. Fulltext Rapid diagnosis of leptospirosis by multiplex PCR
    Ahmed SA, Sandai DA, Musa S, Hoe CH, Riadzi M, Lau KL, et al.
    Malays J Med Sci, 2012 Jul;19(3):9-16.
    PMID: 23610544 MyJurnal
    Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  19. Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication
    Aida AA, Che Man YB, Wong CM, Raha AR, Son R
    Meat Sci, 2005 Jan;69(1):47-52.
    PMID: 22062638 DOI: 10.1016/j.meatsci.2004.06.020
    A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.
    Matched MeSH terms: Polymerase Chain Reaction
  20. Fulltext Evidence of West Nile virus infection in migratory and resident wild birds in west coast of peninsular Malaysia
    Ain-Najwa MY, Yasmin AR, Omar AR, Arshad SS, Abu J, Mohammed HO, et al.
    One Health, 2020 Dec;10:100134.
    PMID: 32405525 DOI: 10.1016/j.onehlt.2020.100134
    West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
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