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Displaying publications 261 - 280 of 320 in total

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Fulltext Reduced susceptibility of Malaysian clinical isolates of Burkholderia pseudomallei to ciprofloxacin

Liew FY, Tay ST, Puthucheary SD

Trop Biomed, 2011 Dec;28(3):646-50.
PMID: 22433895 MyJurnal

Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.

Matched MeSH terms: Polymerase Chain Reaction/methods
Fulltext Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples

Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S

Trop Biomed, 2010 Dec;27(3):611-23.
PMID: 21399603 MyJurnal

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Simultaneous detection of methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by multiplex PCR

Thong KL, Lai MY, Teh C SJ, Chua KH

Trop Biomed, 2011 Apr;28(1):21-31.
PMID: 21602765 MyJurnal

A PCR-based assay that can simultaneously detect and differentiate five different types of nosocomial bacterial pathogens was developed. Six pairs of selected primers targeting femA (132 bp) and mecA (310 bp) of methicillin-resistant Staphylococcus aureus, gltA (722 bp) of Acinetobacter baumannii, phoA (903 bp) of Escherichia coli, mdh (364 bp) of Klebsiella pneumoniae and oprL (504 bp) of Pseudomonas aeruginosa were used in this study. The conditions were optimized for the multiplex PCR to ensure specific amplification of the selected targets. Sensitivity and specificity tests were also carried out using a blind test approach on 50 bacterial cultures and resulted in 100% for both positive and negative predictive values.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext The detection and characterization of pathogenic Leptospira and the use of OMPs as potential antigens and immunogens

Gebriel AM, Subramaniam G, Sekaran SD

Trop Biomed, 2006 Dec;23(2):194-207.
PMID: 17322822 MyJurnal

The detection of leptospires in patient blood in the first week of the disease using PCR provides an early diagnostic tool. PCR using two sets of primers (G1/G2 and B64-I/B64-II) tested with samples seeded with 23 leptospiral strains from pathogenic and non-pathogenic strains was able to amplify leptospiral DNA from pathogenic strains only. Of the 39 antibody negative samples collected from patients suspected for leptospirosis, only 1 sample (2.6%) was PCR positive. Using LSSP-PCR, the G2 primers allowed the characterization of Leptopira species to 10 different genetic signatures which may have epidemiological value in determining species involved in outbreaks. Leptospiral outer membrane proteins from three strains were purified and reacted against patients sera and gave rise to different profiles for pathogenic and non-pathogenic strains. Lymphocytes of mice injected with OMPs proliferated and released IFN(-3) when stimulated in vitro using Leptospira OMP as antigens. This suggests that an immune response could be established using leptospiral OMPs as a putative vaccine. OMPs were also used in a Dot-ELISA to detect antibodies against Leptospira pathogens in humans.

Matched MeSH terms: Polymerase Chain Reaction/methods
Telomerase activation and human papillomavirus infection in invasive uterine cervical carcinoma in a set of Malaysian patients

Cheah PL, Looi LM, Ng MH, Sivanesaratnam V

J Clin Pathol, 2002 Jan;55(1):22-6.
PMID: 11825919

AIM: Telomerase activity was studied in invasive uterine cervical carcinoma to assess whether it was activated during cervical malignant transformation and to look for a possible association with human papillomavirus (HPV) infection in a set of Malaysian patients.
METHODS: Histologically confirmed invasive cervical carcinoma and benign cervices were assayed for telomerase activity using a commercial telomerase polymerase chain reaction (PCR) enzyme linked immunosorbent assay kit. The same cases were subjected to PCR detection of HPV using type specific (HPV types 6b, 11, 16, and 18) followed by L1 open reading frame (ORF) consensus primers.
RESULTS: HPV was detected in 18 (13 HPV-16, one HPV-6b, four only L1 ORF) of 20 invasive cervical carcinoma and one (only L1 ORF) of 19 benign cervices. Raised telomerase activity (A(450 nm) > 0.215) was detected in 11 cervical carcinomas, with A(450 nm) ranging between 0.238 and 21.790 (mean, 3.952) in positive squamous carcinomas, whereas A(450 nm) was only 0.222 in the one positive adenosquamous carcinoma. Five of 11 cervical carcinomas in stage I, three of six in stage II, both in stage III, and the only case in stage IV showed telomerase activation. Increased telomerase activity was noted in five of the 12 lymph node negative, five of the seven lymph node status unknown cases, and the one case with presumed lymph node metastasis. Ten of 18 HPV positive and one of two HPV negative cervical carcinomas showed telomerase upregulation.
CONCLUSIONS: Telomerase is activated in invasive cervical carcinoma. Although larger studies are needed, there seems to be no clear association between telomerase upregulation and HPV status, although there is a suggestion of increased telomerase activity in squamous carcinomas and late stage disease.

Matched MeSH terms: Polymerase Chain Reaction/methods
Fulltext Aberrant Methylation of Tumour Suppressor Gene ADAM12 in Chronic Lympocytic Leukemia Patients: Application of Methylation Specific-PCR Technique

Mohamad A, Hassan R, Husin A, Johan MF, Sulong S

Asian Pac J Cancer Prev, 2021 Jan 01;22(1):85-91.
PMID: 33507683 DOI: 10.31557/APJCP.2021.22.1.85

OBJECTIVE: Chronic Lymphocytic Leukemia (CLL) is a common leukemia among Caucasians but rare in Asians population. We postulated that aberrant methylation either hypermethylation or partial methylation might be one of the silencing mechanisms that inactivates the tumour suppressor genes in CLL. This study aimed to compare the methylation status of tumour suppressor gene, ADAM12, among CLL patients and normal individuals. We also evaluated the association between methylation of ADAM12 and clinical and demographic characteristics of the participants.
METHODS: A total of 25 CLL patients and 25 normal individuals were recruited in this study. The methylation status of ADAM12 was determined using Methylation-Specific PCR (MSP); whereas, DNA sequencing method was applied for validation of the MSP results.
RESULTS: Among CLL patients, 12 (48%) were partially methylated and 13 (52%) were unmethylated. Meanwhile, 5 (20%) and 20 (80.6%) of healthy individuals were partially methylated and unmethylated, respectively. There was a statistically significant association between the status of methylation at ADAM12 and the presence of CLL (p=0.037).
CONCLUSION: The aberrant methylation of ADAM12 found in this study using MSP assay may provide new exposure to CLL that may improve the gaps involved in genetic epigenetic study in CLL.

Matched MeSH terms: Polymerase Chain Reaction/methods*
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*
Development of nanoparticle-assisted PCR assay in the rapid detection of brain-eating amoebae

Gabriel S, Rasheed AK, Siddiqui R, Appaturi JN, Fen LB, Khan NA

Parasitol Res, 2018 Jun;117(6):1801-1811.
PMID: 29675682 DOI: 10.1007/s00436-018-5864-0

Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.

Matched MeSH terms: Polymerase Chain Reaction/methods*
RNomic identification and evaluation of npcTB_6715, a non-protein-coding RNA gene as a potential biomarker for the detection of Mycobacterium tuberculosis

Kanniappan P, Ahmed SA, Rajasekaram G, Marimuthu C, Ch'ng ES, Lee LP, et al.

J Cell Mol Med, 2017 10;21(10):2276-2283.
PMID: 28756649 DOI: 10.1111/jcmm.13148

Technological advances in RNA biology greatly improved transcriptome profiling during the last two decades. Besides the discovery of many small RNAs (sRNA) that are involved in the physiological and pathophysiological regulation of various cellular circuits, it becomes evident that the corresponding RNA genes might also serve as potential biomarkers to monitor the progression of disease and treatment. sRNA gene candidate npcTB_6715 was previously identified via experimental RNomic (unpublished data), and we report its application as potential biomarker for the detection of Mycobacterium tuberculosis (MTB) in patient samples. For proof of principle, we developed a multiplex PCR assay and report its validation with 500 clinical cultures, positive for Mycobacteria. The analysis revealed 98.9% sensitivity, 96.1% specificity, positive and negative predictive values of 98.6% and 96.8%, respectively. These results underscore the diagnostic value of the sRNA gene as diagnostic marker for the specific detection of MTB in clinical samples. Its successful application and the general ease of PCR-based detection compared to standard bacterial culture techniques might be the first step towards 'point-of-care' diagnostics of Mycobacteria. To the best of our knowledge, this is the first time for the design of diagnostic applications based on sRNA genes, in Mycobacteria.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods
Fulltext Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

Gholami K, Loh SY, Salleh N, Lam SK, Hoe SZ

PLoS One, 2017;12(6):e0176368.
PMID: 28591185 DOI: 10.1371/journal.pone.0176368

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
High Prevalence of Human Papillomavirus Types 56 and 70 Identified in the Native Populations of Sabah, Malaysia

Sainei NE, Kumar VS, Chin YS, Salih FAM

Asian Pac J Cancer Prev, 2018 Oct 26;19(10):2807-2813.
PMID: 30360610

Background: Cervical cancer is currently the third most common female cancer in Malaysia , with the human
papillomavirus (HPV) considered as one of the important contributory factors. This study was conducted to determine
HPV prevalence, its genotype distribution, and other potential risk factors among women in Kota Kinabalu, Sabah
in order to evaluate the likely efficacy of current HPV vaccines in the local population. Methods: A total of 240
cervical samples were collected and subjected to DNA extraction, PCR amplification using the MY09/MY11 primer
pair, and restriction fragment length polymorphism (RFLP) for HPV detection and genotyping. Sociodemographic,
clinical, and behavioural data were also collected via questionnaires. Results: The prevalence of HPV infection was
9.6%. The most common HPVs among 13 genotypes were high-risk HPV-56 (16.7%) and probable high-risk HPV-70
(16.7%) followed by HPV-16, -58, -53, -61, -33, -59, and -66 (in decreasing order of prevalence) including the rare
genotypes: HPV-62, -81, -82 and -84. Statistical analyses using logistic regression models showed that HPV infection
was significantly associated with employment (OR 4.94; CI 1.58-15.40) and education at secondary/high school level
(OR 0.13; CI 0.03-0.62). Conclusion: Distribution of HPV genotypes in Sabah indicated a high prevalence of HPV-56
and -70 which are among the rare HPV types in West Malaysia and merit consideration in future strategies for HPV
vaccination specifically for local Sabahan women.

Matched MeSH terms: Polymerase Chain Reaction/methods
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*
HLA-DR2 haplotypic diversity in populations of South-East Asia, northern China, Melanesia and Australian aborigines using PCR-RFLP for DRB1, DRB5, DQA1 and DQB1. A novel DRB1 allele: DRB1*16022

Trejaut J, Bhatia K, Greville WD, Hu KR, Duraisamy G, Nuchprayoon C, et al.

Eur. J. Immunogenet., 1996 Dec;23(6):437-49.
PMID: 8971541

The polymorphism of the human leucocyte antigen HLA-DR2 and the heterogeneity of HLA-DR2 class II-related haplotypes (HLA-DRB1-DRB5-DQA1-DQB1) were investigated in four populations of east and south-east Asia (SEA) and five Melanesian populations using TaqI restriction fragment length polymorphism (RFLP) analysis, and the polymerase chain reaction (PCR) amplification-based techniques PCR-RFLP and sequence-specific oligonucleotide (SSO) typing. The haplotype DRB1*1502-DRB5*0101-DQA1*0102-DQB1*0601 was common in Malaysians, Javanese, Thursday Islanders, Madang, Goroka and the Australian Aborigines, while DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502 was common in the Thai and Thursday Islanders. DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602 was present at a high frequency in Northern Chinese, Goroka, Watut and Australian Aborigines. The study describes four rare or unusual haplotypes: HLA-DRB1*1501-DRB5*0101-DQA1*0101-DQB1*0601, DRB1*1502-DRB5*0101-DQA1*0101-DQB1*0502, DRB1*1502-DRB5*0102-DQA1* 0102-DQB1*0502 and DRB1*1501-DRB5*0101-DQA1*0101/2-DQB1*0503; the latter two were confirmed by segregation in two Javanese families. A new DR2 allele, initially detected by PCR-RFLP and confirmed by DNA sequencing as DRB1*16022 (previously designated DRB1*16Madang), was seen in a Madang individual. A new HLA-DR2 TaqI RFLP subtype, locally designated as DR15U, is also described. This RFLP subtype segregated in a Javanese family and correlated with a typically SEA haplotype, DRB1*1502-DRB5*0102-DQA1*0101-DQB1*0501. The allele HLA-DR16Thai, determined by TaqI DRB RFLP, was found by PCR-RFLP and SSO typing to correlate with a unique SEA haplotype, HLA-DRB1*16021-DRB5*0101-DQA1*0102-DQB1*0502, and was observed in the Thai, Malaysian, Thursday Islander, Javanese and Northern Chinese populations.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Amycolatopsis saalfeldensis sp. nov., a novel actinomycete isolated from a medieval alum slate mine

Carlsohn MR, Groth I, Tan GYA, Schütze B, Saluz HP, Munder T, et al.

Int J Syst Evol Microbiol, 2007 Jul;57(Pt 7):1640-1646.
PMID: 17625209 DOI: 10.1099/ijs.0.64903-0

Three actinomycetes isolated from the surfaces of rocks in a medieval slate mine were examined in a polyphasic taxonomic study. Chemotaxonomic and morphological characteristics of the isolates were typical of strains of the genus Amycolatopsis. The isolates had identical 16S rRNA gene sequences and formed a distinct phyletic line towards the periphery of the Amycolatopsis mediterranei clade, being most closely related to Amycolatopsis rifamycinica. The organisms shared a wide range of genotypic and phenotypic markers that distinguished them from their closest phylogenetic neighbours. On the basis of these results, a novel species, Amycolatopsis saalfeldensis sp. nov., is proposed. The type strain is HKI 0457(T) (=DSM 44993(T)=NRRL B-24474(T)).

Matched MeSH terms: Polymerase Chain Reaction/methods
Kawasaki disease following coronavirus disease 2019 with prolonged fecal viral shedding

Uda K, Okita K, Soneda K, Taniguchi K, Horikoshi Y

Pediatr Int, 2021 05;63(5):597-599.
PMID: 33278321 DOI: 10.1111/ped.14452
Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Diagnostic tools in childhood malaria

Amir A, Cheong FW, De Silva JR, Lau YL

Parasit Vectors, 2018 01 23;11(1):53.
PMID: 29361963 DOI: 10.1186/s13071-018-2617-y

Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
Fulltext Isolation of monoclonal antibodies-escape variant of dengue virus serotype 1

Chua SK, Selvanesan S, Sivalingam B, Chem YK, Norizah I, Zuridah H, et al.

Singapore Med J, 2006 Nov;47(11):940-6.
PMID: 17075660

During an outbreak from December 2004 to March 2005, 138 isolates of dengue virus were prospectively obtained from acute-phase serum samples of 1,067 patients with the provisional clinical diagnosis of acute dengue illness admitted to the adult wards of Hospital Tengku Ampuan Rahimah, Klang, Malaysia. Of the 138 dengue virus isolates, 87, 11, 24 and 3 were typed as dengue serotypes 1, 2, 3 and 4, respectively, by a commercial dengue virus typing kit using monoclonal antibodies (Mab). 13 dengue virus isolates could not be assigned to any specific serotype by serotyping Mab and molecular typing using dengue-type specific molecular typing primer pairs. We report the associated clinical features and limited molecular genetics of this Mab-escape dengue virus variant.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
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*
Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis

Jarolim P, Palek J, Amato D, Hassan K, Sapak P, Nurse GT, et al.

Proc Natl Acad Sci U S A, 1991 Dec 15;88(24):11022-6.
PMID: 1722314

Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.

Matched MeSH terms: Polymerase Chain Reaction/methods
Characterization of Salmonella serovars by PCR-single-strand conformation polymorphism analysis

Nair S, Lin TK, Pang T, Altwegg M

J Clin Microbiol, 2002 Jul;40(7):2346-51.
PMID: 12089246

PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.

Matched MeSH terms: Polymerase Chain Reaction/methods*

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