Displaying publications 301 - 320 of 320 in total

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  1. Dual variant of Epstein-Barr virus in Hodgkin/Reed-Sternberg cells: single-cell PCR study on latent membrane protein-1 gene
    Kim LH, Peh SC, Poppema S
    Int J Cancer, 2003 Nov 1;107(2):250-5.
    PMID: 12949802
    Isolation of single cells permits analysis of DNA or RNA from individual cells among heterogeneous populations. This technique is particularly useful in the study of classical Hodgkin's lymphoma (cHL) due to the scarcity of H/RS tumor cells among large numbers of reactive leukocytes. In a previous study, we found a high frequency of dual LMP-1 variant (concurrent presence of deleted and nondeleted variants) in cHL from whole-tissue sections. For the present study, we applied a single-cell isolation technique to determine the LMP-1 oncogene variant in EBV-associated H/RS cells. Five cases of EBV-infected cHL, containing nondeleted (n=1), deleted (n=1) and dual infection (n=3) based on whole-tissue section analysis, were selected for study. Paraffin-embedded tissue sections were stained with antibody to LMP-1 and positively stained H/RS cells isolated using a semiautomated micromanipulator. Each isolated single cell was subjected to PCR for amplification of the LMP-1 gene flanking the 30 bp deletion region and Xho1 restriction site. Cases with either nondeleted variant or the deleted variant showed similar LMP-1 variant expression in isolated single H/RS cells. However, 1 of the 3 cases with dual variants showed only the deleted variant in H/RS cells. The other 2 cases showed mixed patterns of deleted, nondeleted and dual LMP-1 variants in isolated single H/RS cells. All cases showed loss of the Xho1 restriction site, with the exception of the case with nondeleted LMP-1. Results of single-H/RS cell analysis of the Xho1 restriction site concur with those of whole-tissue section amplification. A mixed pattern of LMP-1 variants was observed in isolated H/RS cells, and it is speculated that this is due to the accumulation of mutation and deletion events.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  2. Tioman virus, a novel paramyxovirus isolated from fruit bats in Malaysia
    Chua KB, Wang LF, Lam SK, Crameri G, Yu M, Wise T, et al.
    Virology, 2001 May 10;283(2):215-29.
    PMID: 11336547
    A search for the natural host of Nipah virus has led to the isolation of a previously unknown member of the family Paramyxoviridae. Tioman virus (TiV) was isolated from the urine of fruit bats (Pteropus hypomelanus) found on the island of the same name off the eastern coast of peninsular Malaysia. An electron microscopic study of TiV-infected cells revealed spherical and pleomorphic-enveloped viral particles (100--500 nm in size) with a single fringe of embedded peplomers. Virus morphogenesis occurred at the plasma membrane of infected cells and morphological features of negative-stained ribonucleoprotein complexes were compatible with that of viruses in the family Paramyxoviridae. Serological studies revealed no cross-reactivity with antibodies against a number of known Paramyxoviridae members except for the newly described Menangle virus (MenV), isolated in Australia in 1997. Failure of PCR amplification using MenV-specific primers suggested that this new virus is related to but different from MenV. For molecular characterization of the virus, a cDNA subtraction strategy was employed to isolate virus-specific cDNA from virus-infected cells. Complete gene sequences for the nucleocapsid protein (N) and phosphoprotein (P/V) have been determined and recombinant N and V proteins produced in baculovirus. The recombinant N and V proteins reacted with porcine anti-MenV sera in Western blot, confirming the serological cross-reactivity observed during initial virus characterization. The lack of a C protein-coding region in the P/V gene, the creation of P mRNA by insertion of 2-G residues, and the results of phylogenetic analyses all indicated that TiV is a novel member of the genus Rubulavirus.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  3. Detection of amplified int-2/FGF-3 gene in primary breast carcinomas using differential polymerase chain reaction
    Naidu R, Wahab NA, Yadav M, Kutty MK, Nair S
    Int J Mol Med, 2001 Aug;8(2):193-8.
    PMID: 11445874
    Amplification of int-2/FGF-3 gene was investigated by differential polymerase chain reaction (dPCR) in 440 archival primary breast carcinoma tissues. Of these, 23 were comedo ductal carcinoma in situ (DCIS), 18 were non-comedo DCIS, 41 were comedo DCIS with adjacent invasive ductal carcinomas, 19 were non-comedo DCIS with adjacent invasive ductal carcinomas, 270 were invasive ductal carcinomas, 33 were invasive lobular carcinomas, 21 were colloid carcinomas and 15 were medullary carcinomas. Int-2 was amplified in 22% (96/440) of the primary breast carcinomas. It was shown that int-2 was amplified in 13% (3/23) of the comedo DCIS, 17% (7/41) of the comedo DCIS and 29% (12/41) of the adjacent invasive ductal carcinomas, 26% (71/270) of the invasive ductal carcinomas, 18% (6/33) of the invasive lobular carcinomas, 10% (2/21) of the colloid carcinomas and 13% (2/15) of the medullary carcinomas. In contrast, int-2 was not amplified in non-comedo DCIS and invasive ductal carcinomas with adjacent non-comedo DCIS lesions. A significant association was observed between int-2 amplification in the in situ components and adjacent invasive lesion (P<0.05). All tumors with int-2 amplification in the in situ lesions (7/7) also demonstrated same degree of amplification in the adjacent invasive components. However, 9% (5/53) of the tumors with no amplified int-2 gene in the in situ components showed int-2 amplification in the adjacent invasive lesions. A significant relationship was noted between amplification of int-2 and lymph node metastases (P<0.05) and poorly differentiated tumors (P<0.05) but not with estrogen receptor status (P>0.05) and proliferation index (Ki-67 and PCNA) (P>0.05). In Malaysia, majority of the patients belong to younger age group (<50 years old) but a comparison of the age groups showed that the amplification of int-2 was not statistically associated with patient age (P>0.05). These observations indicate that amplification of int-2 tends to strengthen the view that int-2 may have the potential to be an indicator of poor prognosis regardless of the age of the patient. Moreover, the presence of int-2 amplification in preinvasive, preinvasive and adjacent invasive lesions, and invasive carcinomas suggest that int-2 could be a marker of genetic instability occurring in early and late stages of tumor development.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  4. Clinico-pathological responses and PCR detection of Corynebacterium pseudotuberculosis and its immunogenic mycolic acid extract in the vital organs of goats
    Odhah MN, Abdullah Jesse FF, Teik Chung EL, Mahmood Z, Haron AW, Mohd Lila MA, et al.
    Microb Pathog, 2019 Oct;135:103628.
    PMID: 31325572 DOI: 10.1016/j.micpath.2019.103628
    Caseous lymphadenitis is an infectious disease of almost all animals, particularly small ruminants that are caused by Corynebacterium pseudotuberculosis. The organism causes the formation of suppurative abscesses in superficial and visceral lymph nodes and in visceral organs. This current study was designed to elucidate the clinicopathological responses and PCR detection of the aetiological agent in the vital organs of goats challenged with C. pseudotuberculosis and its immunogenic mycolic acid extract. A total of twelve clinically healthy crossbred Boer female goats were divided into three groups: A, B, and C (four goats per group). Group A was inoculated intradermally with 2 ml of sterile phosphate buffered saline (PBS) pH 7 as a control group. Group B was inoculated intradermally with 2 ml of mycolic acid extract (1 g/ml), while group C was inoculated intradermally with 2 ml of 10⁹ colony-forming unit (cfu) of live C. pseudotuberculosis. The experimental animals were observed for clinical responses for 90 days post-inoculation and the clinical signs were scored according to the severity. The clinical signs observed in this study were temperature, heart rate, respiratory rate, rumen motility, enlargement of lymph nodes, and body condition score. The experimental animals were euthanised and tissue samples from different anatomical regions of the vital organs were collected in 10% buffered formalin, processed, sectioned, and stained with H&E. Results of both C. pseudotuberculosis and mycolic acid treated groups indicated a significant difference (p 
    Matched MeSH terms: Polymerase Chain Reaction/methods
  5. Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)
    Liu W, Wang YT, Tian DS, Yin ZC, Kwang J
    Dis Aquat Organ, 2002 Apr 24;49(1):11-8.
    PMID: 12093036
    The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  6. A preliminary study of dengue infection in Brunei
    Osman O, Fong MY, Devi S
    Jpn J Infect Dis, 2007 Jul;60(4):205-8.
    PMID: 17642533
    The purpose of this study was to examine the extent of dengue infection in Brunei and to determine the predominant serotype circulating in the country. The study generated useful epidemiological data on dengue infection in Brunei. A total of 271 samples from patients suspected of having dengue infections were selected and analyzed. All patients were seen in clinics and hospitals in Brunei. The samples were collected from April 2005 to April 2006 and transported to the WHO Collaborating Centre for Arbovirus Reference and Research, University of Malaya, Malaysia. The following tests were used to achieve the objectives: in-house IgM-capture enzyme-linked immunosorbent assay, virus isolation in mosquito albopictus cell line (C6/36), and viral RNA detection and serotyping by reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that 45 people were positive for dengue-specific IgM (27 males and 18 females), while RT-PCR detected dengue viral RNA in 12 patients, 3 identified as DEN-1 and 9 as DEN-2. Dengue virus was isolated from 6 patients using the C6/36 cell line; 3 were DEN-2 isolates and 3 were DEN-1 isolates. These data show that dengue virus is circulating in Brunei and the predominant infecting serotype for that period was DEN-2 followed by DEN-1. This study is the first to report the detection and isolation of dengue virus from Brunei using RT-PCR and culture in the C6/36 albopictus mosquito cell line.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
  7. Rapid detection of prognostically important childhood acute lymphoblastic leukemia chimeric transcripts using multiplex SYBR green real-time reverse transcription PCR
    Ibrahim K, Daud SS, Seah YL, Yeoh AE, Ariffin H, Malaysia-Singapore Leukemia Study Group
    Ann Clin Lab Sci, 2008;38(4):338-43.
    PMID: 18988926
    Childhood acute lymphoblastic leukaemia (ALL) is a heterogenous disease in which oncogene fusion transcripts are known to influence the biological behaviour of the different ALL subtypes. Screening for prognostically important transcripts is an important diagnostic step in treatment stratification and prognostication of affected patients. We describe a SYBR-Green real-time multiplex PCR assay to screen for transcripts TEL-AML1, E2A-PBX1, MLL-AF4, and the two breakpoints of BCR-ABL (p190 and p210). Validation of the assay was based on conventional karyotyping results. This new assay provides a rapid, sensitive, and accurate detection method for prognostically important transcripts in childhood ALL.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  8. Evaluation of PCR-RFLP analysis targeting hsp65 and rpoB genes for the typing of mycobacterial isolates in Malaysia
    Ong CS, Ngeow YF, Yap SF, Tay ST
    J Med Microbiol, 2010 Nov;59(Pt 11):1311-1316.
    PMID: 20688949 DOI: 10.1099/jmm.0.021139-0
    In this study, PCR-RFLP analysis (PRA) targeting hsp65 and rpoB gene regions was evaluated for the identification of mycobacterial species isolated from Malaysian patients. Overall, the hsp65 PRA identified 92.2 % of 90 isolates compared to 85.6 % by the rpoB PRA. With 47 rapidly growing species, the hsp65 PRA identified fewer (89.4 %) species than the rpoB PRA (95.7 %), but with 23 slow-growing species the reverse was true (91.3 % identification by the hsp65 PRA but only 52.5 % by the rpoB PRA). There were 16 isolates with discordant PRA results, which were resolved by 16S rRNA and hsp65 gene sequence analysis. The findings in this study suggest that the hsp65 PRA is more useful than the rpoB PRA for the identification of Mycobacterium species, particularly with the slow-growing members of the genus. In addition, this study reports 5 and 12 novel restriction patterns for inclusion in the hsp65 and rpoB PRA algorithms, respectively.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  9. High frequency of germinal centre derivation in diffuse large B cell lymphoma from Asian patients
    Shia AK, Gan GG, Jairaman S, Peh SC
    J Clin Pathol, 2005 Sep;58(9):962-7.
    PMID: 16126878
    Recent reports have divided diffuse large B cell lymphoma (DLBCL) into germinal centre B cell-like and activated B cell-like subgroups with implicated differences in prognosis.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  10. Clinical implications of conventional cytogenetics, fluorescence in situ hybridization (FISH) and molecular testing in chronic myeloid leukaemia patients in the tyrosine kinase inhibitor era - A review
    Ankathil R, Ismail SM, Mohd Yunus N, Sulong S, Husin A, Abdullah AD, et al.
    Malays J Pathol, 2020 Dec;42(3):307-321.
    PMID: 33361712
    Chronic myeloid leukaemia (CML) provides an illustrative disease model for both molecular pathogenesis of cancer and rational drug therapy. Imatinib mesylate (IM), a BCR-ABL1 targeted tyrosine kinase inhibitor (TKI) drug, is the first line gold standard drug for CML treatment. Conventional cytogenetic analysis (CCA) can identify the standard and variant Philadelphia (Ph) chromosome, and any additional complex chromosome abnormalities at diagnosis as well as during treatment course. Fluorescence in situ hybridization (FISH) is especially important for cells of CML patients with inadequate or inferior quality metaphases or those with variant Ph translocations. CCA in conjunction with FISH can serve as powerful tools in all phases of CML including the diagnosis, prognosis, risk stratification and monitoring of cytogenetic responses to treatment. Molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) is used for the detection of BCR-ABL1 transcripts at diagnosis whereas quantitative reverse transcriptase-polymerase chain reaction (qRTPCR) is used at the time of diagnosis as well as during TKI therapy for the quantitation of BCR-ABL1 transcripts to evaluate the molecular response and minimal residual disease (MRD). Despite the excellent treatment results obtained after the introduction of TKI drugs, especially Imatinib mesylate (IM), resistance to TKIs develops in approximately 35% - 40% of CML patients on TKI therapy. Since point mutations in BCR-ABL1 are a common cause of IM resistance, mutation analysis is important in IM resistant patients. Mutations are reliably detected by nested PCR amplification of the translocated ABL1 kinase domain followed by direct sequencing of the entire amplified kinase domain. The objective of this review is to highlight the importance of regular and timely CCA, FISH analysis and molecular testing in the diagnosis, prognosis, assessment of therapeutic efficacy, evaluation of MRD and in the detection of BCR-ABL1 kinase mutations which cause therapeutic resistance in adult CML patients.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
  11. Fulltext Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development
    Dzaki N, Azzam G
    PLoS One, 2018;13(3):e0194664.
    PMID: 29554153 DOI: 10.1371/journal.pone.0194664
    Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.
    Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
  12. Multiplex PCR for detection of Helicobacter pylori infection in gastric biopsies with lower inflammatory score
    Fadilah N, Hanafiah A, Razlan H, Wong ZQ, Mohamed Rose I, Rahman MM
    Br J Biomed Sci, 2016 Oct;73(4):180-187.
    PMID: 27922429
    BACKGROUND: No gold standard has yet been established for the diagnosis of H. pylori infection. A multiplex polymerase chain reaction (mPCR) was developed in this study for rapid, sensitive and specific detection of H. pylori from gastric biopsies.

    METHODS: H. pylori infections were determined by in-house rapid urease test (iRUT), culture, histology and multiplex PCR.

    RESULTS: A total of 140 (60.9%) from 230 patients were positive for H. pylori infection. H. pylori were detected in 9.6% (22/230), 17% (39/230), 12.6% (29/230) and 60% (138/230) of biopsy specimens by culture, iRUT, histology and mPCR, respectively. mPCR identified H. pylori infection in 100% of biopsies with positive histology and culture. All biopsies with positive iRUT yielded positive PCR except two cases. mPCR also detected H. pylori in additional 116, 101 and 109 biopsies that were negative by culture, iRUT and histology, respectively. Positive samples by mPCR showed lower average in H. pylori density, activity and inflammation scores. The Indians showed the highest prevalence of H. pylori infection compared to the Chinese and the Malays. In addition, Chinese patients with older age were significantly infected compared to other ethnicities.

    CONCLUSION: PCR was able to detect the highest numbers of positive cases although the lowest average scores were recorded in the activity, inflammatory and H. pylori density.

    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  13. Fulltext Feasibility of T-cell receptor gamma (TCRgamma) gene rearrangement on formalin-fixed, paraffin-embedded tissues by PCR assays
    Tai YC, Peh SC
    Singapore Med J, 2003 May;44(5):250-5.
    PMID: 13677361
    T- and B-lymphocytes are involved in recognition of foreign antigen by the specificity of their surface T-cell receptor and immunoglobulin, generated by gene rearrangement. Each T- and B-lymphocyte carries unique rearranged TCR or immunoglobulin gene, which has been applied to detect clonal from non-clonal T- and B-cell proliferation.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  14. A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species
    Yean CY, Yin LS, Lalitha P, Ravichandran M
    BMC Microbiol, 2007 Dec 11;7:112.
    PMID: 18070365
    BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.

    RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.

    CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.

    Matched MeSH terms: Polymerase Chain Reaction/methods*
  15. Fulltext Pentaplex PCR assay for detection of hemorrhagic bacteria from stool samples
    Al-Talib H, Latif B, Mohd-Zain Z
    J Clin Microbiol, 2014 Sep;52(9):3244-9.
    PMID: 24958797 DOI: 10.1128/JCM.00891-14
    Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10(3) CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.
    Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
  16. Survey of campylobacter, salmonella and mycoplasmas in house crows (Corvus splendens) in Malaysia
    Ganapathy K, Saleha AA, Jaganathan M, Tan CG, Chong CT, Tang SC, et al.
    Vet Rec, 2007 May 05;160(18):622-4.
    PMID: 17483380
    House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  17. Fulltext A novel and non-invasive approach utilising nasal washings for the detection of nasopharyngeal carcinoma
    Tan GW, Sivanesan VM, Abdul Rahman FI, Hassan F, Hasbullah HH, Ng CC, et al.
    Int J Cancer, 2019 Oct 15;145(8):2260-2266.
    PMID: 30698824 DOI: 10.1002/ijc.32173
    Nasopharyngeal carcinoma (NPC) is an epithelial cancer of the nasopharynx which is highly associated with Epstein-Barr virus (EBV). Worldwide, most of the top 20 countries with the highest incidence and mortality rates of NPC are low- and middle-income countries. Many studies had demonstrated that EBV could be detected in the tissue, serum and plasma of NPC patients. In this study, we explored the potential of assays based on non-invasive nasal washings (NW) as a diagnostic and prognostic tool for NPC. A total of 128 patients were evaluated for NW EBV DNA loads and a subset of these samples were also tested for 27 EBV and human miRNAs shortlisted from literature. EBV DNA and seven miRNAs showed area under the receiver operating characteristic curve (AUC) values of more than 0.7, suggestive of their potential utility to detect NPC. Logistic regression analyses suggested that combination of two NW assays that test for EBNA-1 and hsa-miR-21 had the best performance in detecting NPC. The trend of NW EBV DNA load matched with clinical outcome of 71.4% (10 out of 14) NPC patients being followed-up. In summary, the non-invasive NW testing panel may be particularly useful for NPC screening in remote areas where healthcare facilities and otolaryngologists are lacking, and may encourage frequent testing of individuals in the high risk groups who are reluctant to have their blood tested. However, further validation in an independent cohort is required to strengthen the utility of this testing panel as a non-invasive detection tool for NPC.
    Matched MeSH terms: Polymerase Chain Reaction/methods
  18. Fulltext Allele-specific polymerase chain reaction for the detection of Alzheimer's disease-related single nucleotide polymorphisms
    Darawi MN, Ai-Vyrn C, Ramasamy K, Hua PP, Pin TM, Kamaruzzaman SB, et al.
    BMC Med Genet, 2013;14:27.
    PMID: 23419238 DOI: 10.1186/1471-2350-14-27
    The incidence of Alzheimer's disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  19. Isolation and PCR detection of rickettsiae from clinical and rodent samples in Malaysia
    Tay ST, Rohani MY, Ho TM, Devi S
    Southeast Asian J Trop Med Public Health, 2002 Dec;33(4):772-9.
    PMID: 12757225
    Isolation of rickettsiae from patients' blood samples and organ samples of wild rodents from areas with high seroprevalence of rickettsial infections was attempted using cell culture assay and animal passages. L929 mouse fibroblast cells grown in 24 well tissue culture plate were inoculated with buffy coat of febrile patients and examined for the growth of rickettsiae by Giemsa, Gimenez staining and direct immunofluorescence assay. No rickettsiae were isolated from 48 patients' blood samples. No symptomatic infections were noted in mice or guinea pigs infected with 50 organ samples of wild rodents. There was no rickettsial DNA amplified from these samples using various PCR detection systems for Orientia tsutsugamushi, typhus and spotted fever group rickettsiae.
    Matched MeSH terms: Polymerase Chain Reaction/methods*
  20. Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens
    Rasool NB, Monroe SS, Glass RI
    J Virol Methods, 2002 Feb;100(1-2):1-16.
    PMID: 11742648
    Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol-chloroform-isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol-chloroform-isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol-chloroform-isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate 'universal' nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol-chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a 'universal' nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.
    Matched MeSH terms: Polymerase Chain Reaction/methods
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