Displaying publications 41 - 60 of 305 in total

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  1. Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs, burgers, frankfurters and traditional Chinese herbal jelly powder
    Asing, Ali E, Hamid SB, Hossain M, Ahamad MN, Hossain SM, et al.
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2016 Nov;33(11):1643-1659.
    PMID: 27643977
    The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25-153.00%, PCR efficiency of 99-100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50-7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32-28.90 ± 0.42) and melting curves (74.63-78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction*
  2. Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)
    Atshan SS, Shamsudin MN, Karunanidhi A, van Belkum A, Lung LT, Sekawi Z, et al.
    Infect Genet Evol, 2013 Aug;18:106-12.
    PMID: 23669446 DOI: 10.1016/j.meegid.2013.05.002
    Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  3. Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm
    Atshan SS, Shamsudin MN, Lung LT, Ling KH, Sekawi Z, Pei CP, et al.
    Gene, 2012 Feb 25;494(2):219-24.
    PMID: 22222139 DOI: 10.1016/j.gene.2011.12.010
    The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  4. Polymerase chain reaction cycle threshold value as prerequisite for reporting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection
    Awang H, Hamzah FH, Ahmad MH, Mahmood MF, Wahab A, Embong K, et al.
    Infect Dis (Lond), 2021 05;53(5):390-392.
    PMID: 33512265 DOI: 10.1080/23744235.2021.1876913
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  5. Fulltext Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice
    Azizi P, Rafii MY, Abdullah SN, Hanafi MM, Maziah M, Sahebi M, et al.
    Front Plant Sci, 2016;7:773.
    PMID: 27379107 DOI: 10.3389/fpls.2016.00773
    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48, and 72 h after inoculation in transgenic plants, while it was increased in the inoculated control plants. This study successfully clarified that over-expression of the Pikh gene in transgenic plants can improve their blast resistance against the M. oryzae pathotype P7.2.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  6. Fulltext Prevalence of uridine glucuronosyl transferase 1A1 (UGT1A1) mutations in Malay neonates with severe jaundice
    Azlin I, Wong FL, Ezham M, Hafiza A, Ainoon O
    Malays J Pathol, 2011 Dec;33(2):95-100.
    PMID: 22299209 MyJurnal
    A number of genetic risk factors have been implicated in the development of neonatal severe hyperbilirubinaemia. This includes mutations in the uridine glucoronosyl transferase 1A1 (UGT1A1) gene which is responsible for unconjugated hyperbilirubinemia in Gilbert's Syndrome. We studied the prevalence of UGT1A1 gene mutations in a group of Malay neonates to determine whether they are risk factors to severe neonatal jaundice. One hundred and twenty-five Malay neonates with severe hyperbilirubinemia were studied. Ninety-eight infants without severe hyperbilirubinaemia were randomly selected from healthy Malay term infants (controls). DNA from EDTA cord blood samples were examined for UGT1A1 mutations nt211G > A and nt247T > C using established Taqman SNP genotyping assays and the UGT1A1*28 variant was detected by the Agilent 2100 bioanalyzer. All samples were also screened for common Malay G6PD variants using established techniques. The frequency of UGT1A1 211G > A mutation is significantly higher in the severely hyperbilirubinemic group (13%) than the control group (4%; p = 0.015) and all the positive cases were heterozygous for the mutation. There was no significant difference in the frequency of UGT1A1*28 mutation between the severely hyperbilirubinemic (3.5%) and the control group (0.01%; p = 0.09). None of the neonates in both groups carried the nt247 T > C mutation. The prevalence of G6PD mutation was significantly higher in the severely jaundiced group than control (9% vs 4%; p = 0.04). In conclusion, nt 211 G > A alleles constitute at least 12% of UGT1A1 mutations underlying unconjugated hyperbilirubinemia and appears to be a significant independent risk factor associated with severe neonatal hyperbilirubinemia in the Malay newborns.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  7. U6 snRNA is a suitable endogenous control for microRNA-124 and -134 in cultured rat hippocampal neurons
    Azlina Ahmad-Annuar, Ai Sze Ching
    Sains Malaysiana, 2015;44:1481-1488.
    As researchers seek to determine the cellular mechanisms underlying biological processes, they have turned to analyze the functional role of microRNAs to understand this process in details. Here, we investigated the expression pattern of two microRNAs, miR-124 and -134 in maturing neurons and found that the choice of endogenous controls influenced the observed expression levels of these microRNAs. We have cultured rat hippocampal neurons and performed quantitative PCR on the microRNAs using Taqman gene expression assays. The expression of miRNAs was normalised with selected endogenous controls. Using BestKeeper and NormFinder software, we found that 18S rRNA and 5S rRNA to be unsuitable as endogenous controls in this system, while normalising to U6 snRNA produced more consistent results. Our study would like to highlight the importance of empirically testing proposed endogenous controls in any model system before data interpretation is carried out.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  8. Fulltext Gene Polymorphisms of the Renin-Angiotensin-Aldosterone System as Risk Factors for the Development of In-Stent Restenosis in Patients with Stable Coronary Artery Disease
    Azova M, Timizheva K, Ait Aissa A, Blagonravov M, Gigani O, Aghajanyan A, et al.
    Biomolecules, 2021 05 20;11(5).
    PMID: 34065198 DOI: 10.3390/biom11050763
    This study investigated the renin-angiotensin-aldosterone system (RAAS) gene polymorphisms as possible genetic risk factors for the restenosis development in patients with drug-eluting stents. 113 participants had coronary artery disease and underwent stenting. The control group consisted of 62 individuals with intact coronary arteries. Patients were divided into two groups: with in-stent restenosis (ISR) and without it. The patients with ISR were classified into subgroups by the terms of the restenosis development and age. Real-time PCR and Restriction Fragment Length Polymorphism-PCR were used to genotype the study participants for RAAS gene polymorphisms. We found that the development of restenosis is generally associated with the minor A allele for renin (REN) rs2368564 and the major TT genotype for angiotensinogen (AGT) rs699. The heterozygous genotype for AGT rs4762 acts as a protective marker. A minor A allele for angiotensin II type 2 receptor (AGTR2) rs1403543 is associated with a risk of restenosis in people under 65 years old. Among patients with the early ISR, heterozygotes for angiotensin II type 1 receptor (AGTR1) rs5186 are more frequent, as well as A allele carriers for AGTR2 rs1403543. A minor homozygous genotype for REN rs41317140 and heterozygous genotype for aldosterone synthase (CYP11B2) rs1799998 are predisposed to the late restenosis. Thus, to choose the effective treatment tactics for patients with coronary artery disease, it is necessary to genotype patients for the RAAS polymorphisms, which, along with age and clinical characteristics, will allow a comprehensive assessment of the risk of the restenosis development after stenting.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  9. In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism
    Bakri MM, Rich AM, Cannon RD, Holmes AR
    Mol Oral Microbiol, 2015 Feb;30(1):27-38.
    PMID: 24975985 DOI: 10.1111/omi.12064
    Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  10. Direct detection of Strongyloides infection via molecular and antigen detection methods
    Balachandra D, Ahmad H, Arifin N, Noordin R
    Eur J Clin Microbiol Infect Dis, 2021 Jan;40(1):27-37.
    PMID: 32729057 DOI: 10.1007/s10096-020-03949-x
    Laboratory diagnosis of Strongyloides infections can be grouped into direct and indirect detection methods, and a combination of the two methods is often needed to reach an accurate and timely diagnosis. This review focuses on non-conventional direct detection via molecular and antigen detection assays. Conventional PCR is the most commonly used molecular diagnostic for Strongyloides. Real-time PCR is accurate and highly sensitive for quantitative and qualitative analysis. Meanwhile, PCR-RFLP can efficiently distinguish human and dog isolates of S. stercoralis, S. fuelleborni (from monkey), and S. ratti (from rodent). Loop-mediated isothermal amplification (LAMP) amplifies DNA isothermally with high specificity, efficiency, and rapidity, and has potential for point-of-care (POC) translation. As for antigen detection assay, coproantigen detection ELISAs for strongyloidiasis traditionally relied on raising rabbit polyclonal antibodies against the parasite antigens for use as capture or detection reagents. Subsequently, hybridoma technology using animals has enabled the discovery of monoclonal antibodies specific to Strongyloides antigens and was utilised to develop antigen detection assays. In recent times, phage display technology has facilitated the discovery of scFv antibody against Strongyloides protein that can accelerate the development of such assays. Improvements in both direct detection methods are being made. Strongyloides molecular diagnostics is moving from the detection of a single infection to the simultaneous detection of soil-transmitted helminths. Meanwhile, antigen detection assays can also be multiplexed and aptamers can be used as antigen binders. In the near future, these two direct detection methods may be more widely used as diagnostic tools for strongyloidiasis.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  11. Fulltext Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
    Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.
    Virol J, 2020 Oct 27;17(1):164.
    PMID: 33109247 DOI: 10.1186/s12985-020-01436-5
    BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy.

    METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.

    RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P 

    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  12. Fulltext Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR
    Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R
    Trop Biomed, 2012 Sep;29(3):434-42.
    PMID: 23018507
    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  13. Decolourisation of Acid Orange 7 recalcitrant auto-oxidation coloured by-products using an acclimatised mixed bacterial culture
    Bay HH, Lim CK, Kee TC, Ware I, Chan GF, Shahir S, et al.
    Environ Sci Pollut Res Int, 2014 Mar;21(5):3891-906.
    PMID: 24293297 DOI: 10.1007/s11356-013-2331-4
    This study focuses on the biodegradation of recalcitrant, coloured compounds resulting from auto-oxidation of Acid Orange 7 (AO7) in a sequential facultative anaerobic-aerobic treatment system. A novel mixed bacterial culture, BAC-ZS, consisting of Brevibacillus panacihumi strain ZB1, Lysinibacillus fusiformis strain ZB2, and Enterococcus faecalis strain ZL bacteria were isolated from environmental samples. The acclimatisation of the mixed culture was carried out in an AO7 decolourised solution. The acclimatised mixed culture showed 98 % decolourisation within 2 h of facultative anaerobic treatment using yeast extract and glucose as co-substrate. Subsequent aerobic post treatment caused auto-oxidation reaction forming dark coloured compounds that reduced the percentage decolourisation to 73 %. Interestingly, further agitations of the mixed culture in the solution over a period of 48 h significantly decolourise the coloured compounds and increased the decolourisation percentage to 90 %. Analyses of the degradation compounds using UV-visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR) and high performance liquid chromatography (HPLC) showed complete degradation of recalcitrant AO7 by the novel BAC-ZS. Phytotoxicity tests using Cucumis sativus confirmed the dye solution after post aerobic treatment were less toxic compared to the parent dye. The quantitative real-time PCR revealed that E. faecalis strain ZL was the dominant strain in the acclimatised mix culture.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  14. Fulltext Identification of Reference Genes in Chicken Intraepithelial Lymphocyte Natural Killer Cells Infected with Very-virulent Infectious Bursal Disease Virus
    Boo SY, Tan SW, Alitheen NB, Ho CL, Omar AR, Yeap SK
    Sci Rep, 2020 05 22;10(1):8561.
    PMID: 32444639 DOI: 10.1038/s41598-020-65474-3
    Due to the limitations in the range of antibodies recognising avian viruses, quantitative real-time PCR (RT-qPCR) is still the most widely used method to evaluate the expression of immunologically related genes in avian viruses. The objective of this study was to identify suitable reference genes for mRNA expression analysis in chicken intraepithelial lymphocyte natural killer (IEL-NK) cells after infection with very-virulent infectious bursal disease virus (vvIBDV). Fifteen potential reference genes were selected based on the references available. The coefficient of variation percentage (CV%) and average count of these 15 genes were determined by NanoString technology for control and infected samples. The M and V values for shortlisted reference genes (ACTB, GAPDH, HMBS, HPRT1, SDHA, TUBB1 and YWHAZ) were calculated using geNorm and NormFinder. GAPDH, YWHAZ and HMBS were the most stably expressed genes. The expression levels of three innate immune response related target genes, CASP8, IL22 and TLR3, agreed in the NanoString and RNA sequencing (RNA-Seq) results using one or two reference genes for normalisation (not HMBS). In conclusion, GAPDH and YWHAZ could be used as reference genes for the normalisation of chicken IEL-NK cell gene responses to infection with vvIBDV.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  15. Occult hepatitis B infection in blood donors from South East Asia: molecular characterisation and potential mechanisms of occurrence
    Candotti D, Lin CK, Belkhiri D, Sakuldamrongpanich T, Biswas S, Lin S, et al.
    Gut, 2012 Dec;61(12):1744-53.
    PMID: 22267593 DOI: 10.1136/gutjnl-2011-301281
    To investigate the molecular basis of occult hepatitis B virus (HBV) infection (OBI) in Asian blood donors.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  16. Autophagy in Helicobacter pylori Infection and Related Gastric Cancer
    Castaño-Rodríguez N, Kaakoush NO, Goh KL, Fock KM, Mitchell HM
    Helicobacter, 2015 Oct;20(5):353-69.
    PMID: 25664588 DOI: 10.1111/hel.12211
    Autophagy, a degradation pathway in which cytoplasmic content is engulfed and degraded by lysosomal hydrolases, plays a pivotal role in infection and inflammation. Given that defects in autophagy lead to increased susceptibility to infection, we investigated the role of autophagy in Helicobacter pylori-related gastric cancer (GC).
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  17. Emergence of Aureobasidium pullulans as human fungal pathogen and molecular assay for future medical diagnosis
    Chan GF, Puad MS, Chin CF, Rashid NA
    Folia Microbiol (Praha), 2011 Sep;56(5):459-67.
    PMID: 21909832 DOI: 10.1007/s12223-011-0070-9
    Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  18. Fulltext Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture
    Chan PL, Rose RJ, Abdul Murad AM, Zainal Z, Low ET, Ooi LC, et al.
    PLoS One, 2014;9(6):e99774.
    PMID: 24927412 DOI: 10.1371/journal.pone.0099774
    The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  19. Fulltext Detection of Tuberculosis (TB) using gold standard method, direct sputum smears microscopy, PCR, qPCR and electrochemical DNA sensor: a mini review
    Che Engku Noramalina Che-Engku-Chik, Siti Sarah Othman, Helmi Wasoh, Nor Azah Yusof, Jaafar Abdullah, Mohd Hazani Mat Zaid
    Journal of Biochemistry, Microbiology and Biotechnology, 2016;4(2):16-21.
    MyJurnal
    Despite the continued effort globally made to control the growing case of Tuberculosis (TB), it
    continues to be regarded as the second deadliest disease after the HIV. There are various
    methods developed to diagnose TB, most of which having the criteria of sensitive, selective,
    cheap and portable to be used in robust applications. Even with the advancement in medication,
    the important keys including early stage diagnosis is yet to be considered. In diagnosing TB, the
    only technique remained as the gold standard method is the culturing method, which is the Acid
    Fast Bacilli (AFB) staining. On the other hand, molecular technique utilising Polymerase Chain
    Reaction (PCR) assay is preferred as a non-culturing method. Additionally, as molecular
    techniques become advanced, real-time PCR or quantitative PCR (qPCR) using multiple probes
    in one shot has raised interest among researchers, because it can skip the process of gel
    electrophoresis. Recently, researchers have been working on electrochemical DNA sensors
    which are sensitive, selective, rapid, cheap and can meet with point of care (POC) testing
    requirements to diagnose TB.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
  20. Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China
    Chen L, Yao XJ, Xu SJ, Yang H, Wu CL, Lu J, et al.
    Arch Virol, 2018 Nov 29.
    PMID: 30498962 DOI: 10.1007/s00705-018-4112-3
    Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction
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