Displaying publications 1 - 20 of 314 in total

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  1. Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction
    Ali ME, Hashim U, Mustafa S, Che Man YB, Dhahi TS, Kashif M, et al.
    Meat Sci, 2012 Aug;91(4):454-9.
    PMID: 22444666 DOI: 10.1016/j.meatsci.2012.02.031
    A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  2. Fulltext A comparative evaluation of dengue diagnostic tests based on single-acute serum samples for laboratory confirmation of acute dengue
    Chua KB, Mustafa B, Abdul Wahab AH, Chem YK, Khairul AH, Kumarasamy V, et al.
    Malays J Pathol, 2011 Jun;33(1):13-20.
    PMID: 21874746
    A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  3. Detection and discrimination of Mycobacterium tuberculosis complex
    Issa R, Mohd Hassan NA, Abdul H, Hashim SH, Seradja VH, Abdul Sani A
    Diagn Microbiol Infect Dis, 2012 Jan;72(1):62-7.
    PMID: 22078904 DOI: 10.1016/j.diagmicrobio.2011.09.021
    A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  4. Identification of the genomic mutation in Epha4(rb-2J/rb-2J) mice
    Mohd-Zin SW, Abdullah NL, Abdullah A, Greene ND, Cheah PS, Ling KH, et al.
    Genome, 2016 Jul;59(7):439-48.
    PMID: 27373307 DOI: 10.1139/gen-2015-0142
    The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4(rb-2J/rb-2J), is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or "hopping gait" phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4(rb-2J) corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4(rb-2J) allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein-protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  5. Selection of reference genes for quantitative studies in acute myeloid leukaemia
    Lee C, Yiau KXS, Lee LJ, Chong PP, Chang KM, Abdullah M
    Malays J Pathol, 2019 Dec;41(3):313-326.
    PMID: 31901916
    INTRODUCTION: Quantitative polymerase chain reaction (qPCR) is commonly used in the investigation of acute myeloid leukaemias (AML). Stable reference genes (RG) are essential for accurate and reliable reporting but no standard method for selection has been endorsed.

    MATERIALS AND METHODS: We evaluated simple statistics and published model-based approaches. Multiplex-qPCR was conducted to determine the expression of 24 candidate RG in AMLs (N=9). Singleplex-qPCR was carried out on selected RG (SRP14, B2M and ATP5B) and genes of interest in AML (N=15) and healthy controls, HC (N=12).

    RESULTS: RG expression levels in AML samples were highly variable and coefficient of variance (CV) ranged from 0.37% to 10.17%. Analysis using GeNorm and Normfinder listed different orders of most stable genes but the top seven (ACTB, UBE2D2, B2M, NF45, RPL37A, GK, QARS) were the same. In singleplex-qPCR, SRP14 maintained the lowest CV in AML samples. B2M, one of most stable reference genes in AML, was expressed near significantly different in AML and HC. GeNorm selected ATP5B+SRP14 while Normfinder chose SRP14+B2M as the best two RG in combination. The median expressions of combined RG genes in AML compared to HC were less significantly different than individually implying smaller expression variation after combination. Genes of interest normalised with RG in combination or individually, displayed significantly different expression patterns.

    CONCLUSIONS: The selection of best reference gene in qPCR must consider all sample sets. Model-based approaches are important in large candidate gene analysis. This study showed combination of RG SRP14+B2M was the most suitable normalisation factor for qPCR analysis of AML and healthy individuals.

    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  6. Fulltext The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen
    Teoh BT, Sam SS, Tan KK, Johari J, Abd-Jamil J, Hooi PS, et al.
    Sci Rep, 2016 06 09;6:27663.
    PMID: 27278716 DOI: 10.1038/srep27663
    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  7. 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
  8. Fulltext A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus
    Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, et al.
    Sci Rep, 2018 12 05;8(1):17632.
    PMID: 30518924 DOI: 10.1038/s41598-018-36043-6
    Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  9. Fulltext Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification
    Teoh BT, Sam SS, Tan KK, Danlami MB, Shu MH, Johari J, et al.
    J Clin Microbiol, 2015 Mar;53(3):830-7.
    PMID: 25568438 DOI: 10.1128/JCM.02648-14
    A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  10. Molecular phylogeographic studies on Paragonimus westermani in Asia
    Iwagami M, Ho LY, Su K, Lai PF, Fukushima M, Nakano M, et al.
    J Helminthol, 2000 Dec;74(4):315-22.
    PMID: 11138020
    The lung fluke, Paragonimus westermani (Kerbert, 1878), is widely distributed in Asia, and exhibits much variation in its biological properties. Previous phylogenetic studies using DNA sequences have demonstrated that samples from north-east Asia form a tight group distinct from samples from south Asia (Philippines, Thailand, Malaysia). Among countries from the latter region, considerable molecular diversity was observed. This was investigated further using additional DNA sequences (partial mitochondrial cytochrome c oxidase subunit 1 (COI) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) from additional samples of P. westermani. Phylogenies inferred from these again found three or four groups within P. westermani, depending on the method of analysis. Populations of P. westermani from north-east Asia use snail hosts of the family Pleuroceridae and differ in other biological properties from populations in south Asia (that use snail hosts of the family Thiaridae). It is considered that the populations we sampled can be divided into two species, one in north-east Asia and the other in south Asia.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  11. Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus
    Hairul Aini H, Omar AR, Hair-Bejo M, Aini I
    Microbiol Res, 2008;163(5):556-63.
    PMID: 16971101
    The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  12. Sequence analysis of Malaysian infectious bursal disease virus isolate and the use of reverse transcriptase nested polymerase chain reaction enzyme-linked immunosorbent assay for the detection of VP2 hypervariable region
    Phong SF, Hair-Bejo M, Omar AR, Aini I
    Avian Dis, 2003 Jan-Mar;47(1):154-62.
    PMID: 12713171
    The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10(-4) dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  13. Rapid detection of the UGT1A1 single nucleotide polymorphism G211A using real-time PCR with Taqman minor groove binder probes
    Wong FL, Wang MK, Boo NY, Hamidah NH, Ainoon BO
    J Clin Lab Anal, 2007;21(3):167-72.
    PMID: 17506482
    The UGT1A1 Taqman MGB probe single nucleotide polymorphism (SNP) genotyping assay was developed to detect nucleotide 211 of the UDP-glucoronocyltransferase 1A1 (UGT1A1) gene. Defects in this enzyme interfere with process of conjugation of bilirubin and cause unconjugated hyperbilirubinemia. Variation at nucleotide 211 in the coding region of the UGT1A1 gene has been shown to be prevalent in Japanese and Chinese. Using an ABI sequence detection system (SDS) 7000, an allele-specific real-time PCR-based genotyping method was established to detect nucleotide G211A. Cord blood from 125 infants without hyperbilirubinemia (controls) were compared with cord blood from 74 infants (cases) with severe hyperbilirubinemia (total serum bilirubin > 300 micromol/L). Homozygous variation of the UGT1A1 gene at nucleotide 211(A/A) is significantly more common in cases (14.9%) than in controls (0.8%) (P<0.001). Direct sequencing from 20 randomly selected samples showed eight samples with homozygous wild type, seven with homozygous variant, and five samples were heterozygous. The result from this assay was in complete concordance with the DNA sequencing result and clearly discriminate wild-type (G/G), homozygous variant (A/A), and heterozygous (G/A). This assay is rapid and robust for screening of SNP G211A to determine if this polymorphism plays a role in causing severe neonatal jaundice in the local context.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  14. Fulltext Prenatal diagnosis of aneuploidies in amniotic fluid by multiple ligation-dependent probe amplification (MLPA) analysis
    Hamidah NH, Munirah AR, Hafiza A, Farisah AR, Shuhaila A, Norzilawati MN, et al.
    Malays J Pathol, 2014 Dec;36(3):163-8.
    PMID: 25500514 MyJurnal
    Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  15. Real-time quantification for BCR-ABL transcripts in chronic myeloid leukaemia patients in UKMMC, Malaysia
    Wong FL, Hamidah NH, Hawa AA, Nurul AN, Leong CF, Saw F, et al.
    Malays J Pathol, 2011 Dec;33(2):107-12.
    PMID: 22299211
    Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*; Real-Time Polymerase Chain Reaction/methods*
  16. Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products
    Hossain MA, Ali ME, Hamid SB, Hossain SM, Asing, Nizar NN, et al.
    Food Chem, 2017 Jun 01;224:97-104.
    PMID: 28159299 DOI: 10.1016/j.foodchem.2016.12.062
    Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  17. Fulltext Development of qPCR platform with probes for quantifying prevalent and biomedically relevant human gut microbial taxa
    Kurina I, Popenko A, Klimenko N, Koshechkin S, Chuprikova L, Filipenko M, et al.
    Mol Cell Probes, 2020 Aug;52:101570.
    PMID: 32304824 DOI: 10.1016/j.mcp.2020.101570
    Nowadays the advent of innovative high-throughput sequencing allows obtaining high-quality microbiome profiling. However, PCR-based tests are still considered the "golden standard" for many clinical applications. Here, we designed a qPCR-based platform with fluorescent-labeled oligonucleotide probes for assessing human gut microbiome composition. The system allows conducting qualitative and semiquantitative analysis for 12 prokaryotic taxa that are prevalent in the human gut and associated with diseases, diet, age and other factors. The platform was validated by comparing microbiome profile data obtained with two different methods - the platform and high-throughput 16S rRNA sequencing - across 42 stool samples. The test can form the basis for precise and cost-efficient microbiome assay for large-scale surveys including clinical trials with interventions related to diet and disease risks.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  18. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget
    Rahman MM, Hamid SB, Basirun WJ, Bhassu S, Rashid NR, Mustafa S, et al.
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2016;33(1):10-8.
    PMID: 26458055 DOI: 10.1080/19440049.2015.1104558
    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
  19. Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells
    Sultana S, Hossain MAM, Naquiah NNA, Ali ME
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Sep;35(9):1662-1673.
    PMID: 30028648 DOI: 10.1080/19440049.2018.1500719
    Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  20. Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers
    Goodwin W, Alimat S
    Electrophoresis, 2017 04;38(7):1007-1015.
    PMID: 28008628 DOI: 10.1002/elps.201600383
    The SNPforID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single-base-extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP-7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP-4™ on ABI 310 PRISM® and polymers POP-4™, POP-6™ and POP-7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP-6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP-7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
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