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A PCR-based sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes

Phua AC, Abdullah RB, Mohamed Z

J. Reprod. Dev., 2003 Aug;49(4):307-11.
PMID: 14967923

Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn x Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.

Matched MeSH terms: DNA Primers/genetics
Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi

Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS

Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
PMID: 27162264 DOI: 10.4269/ajtmh.15-0670

The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.

Matched MeSH terms: DNA Primers/genetics
Fulltext A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi

Divis PC, Shokoples SE, Singh B, Yanow SK

Malar J, 2010 Nov 30;9:344.
PMID: 21114872 DOI: 10.1186/1475-2875-9-344

BACKGROUND: The misdiagnosis of Plasmodium knowlesi by microscopy has prompted a re-evaluation of the geographic distribution, prevalence and pathogenesis of this species using molecular diagnostic tools. In this report, a specific probe for P. knowlesi, that can be used in a previously described TaqMan real-time PCR assay for detection of Plasmodium spp., and Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, was designed and validated against clinical samples.
METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.

Matched MeSH terms: DNA Primers/genetics
A comparison of the VP1, VP2, and VP4 regions for molecular typing of human enteroviruses

Perera D, Shimizu H, Yoshida H, Tu PV, Ishiko H, McMinn PC, et al.

J Med Virol, 2010 Apr;82(4):649-57.
PMID: 20166171 DOI: 10.1002/jmv.21652

The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.

Matched MeSH terms: DNA Primers/genetics
Fulltext A duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains

Benacer D, Zain SNM, Lewis JW, Khalid MKNM, Thong KL

Rev Soc Bras Med Trop, 2017 Mar-Apr;50(2):239-242.
PMID: 28562762 DOI: 10.1590/0037-8682-0364-2016

INTRODUCTION:: This study aimed to develop a duplex endpoint PCR assay for rapid detection and differentiation of Leptospira strains.

METHODS:: Primers were designed to target the rrs (LG1/LG2) and ligB (LP1/LP2) genes to confirm the presence of the Leptospira genus and the pathogenic species, respectively.

RESULTS:: The assay showed 100% specificity against 17 Leptospira strains with a limit of detection of 23.1pg/µl of leptospiral DNA and sensitivity of 103 leptospires/ml in both spiked urine and water.

CONCLUSIONS:: Our duplex endpoint PCR assay is suitable for rapid early detection of Leptospira with high sensitivity and specificity.

Matched MeSH terms: DNA Primers*
A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies

Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA

Am J Trop Med Hyg, 1999 Apr;60(4):687-92.
PMID: 10348249

A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus- or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/microl of blood using DNA prepared from 25-microl blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.

Matched MeSH terms: DNA Primers
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: DNA Primers
Fulltext A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

Al-Talib H, Yean CY, Al-Khateeb A, Hassan H, Singh KK, Al-Jashamy K, et al.

BMC Microbiol, 2009;9:113.
PMID: 19476638 DOI: 10.1186/1471-2180-9-113

Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern.

Matched MeSH terms: DNA Primers
Fulltext A rapid and sensitive Loop-mediated isothermal amplification assay for detection of pork DNA based on porcine tRNA lys and ATPase 8 genes

Abdullahi, U.F., Igwenagu, E., Aliyu, S., Mu’azu, A., Naim, R., Wan-Taib, W.R.

International Food Research Journal, 2017;24(4):1357-1361.
MyJurnal

This study describes the development of a rapid and sensitive Loop-mediated isothermal
amplification assay for detection of swine DNA in adulterated meat and meat products. The
need to protect consumer’s right to eat foods of their choices, has made it imperative for
researchers to develop efficient means of screening and certification of food products. Six sets
of LAMP primers designed based on porcine tRNA lysine gene and ATPase subunit 8 genes
were used for the assay. Amplification was carried out under constant temperature (630C), using
a simple laboratory water bath. Average time spent in amplification and detection of results was
25 min. All results were visually detected and confirmed by electrophoresis. Detection limit of
the assay was 0.03 femtogram (fg) much high than the PCR assay, and detection probability of
the assay was 100%. Detection of 0.5% of pork spiked with 99.5% of cattle beef is indicative
of the sensitivity and robustness of the assay. This could serve as a prototype for development
of a sensitive and inexpensive Swine DNA LAMP detection kit.

Matched MeSH terms: DNA Primers
A simple method for the detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase chain reaction

Zainuddin Z, Teh LK, Suhaimi AW, Salleh MZ, Ismail R

Clin Chim Acta, 2003 Oct;336(1-2):97-102.
PMID: 14500040 DOI: 10.1016/s0009-8981(03)00319-x

BACKGROUND: Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles.
METHOD: Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results.
RESULTS: We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence.
CONCLUSION: This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.

Matched MeSH terms: DNA Primers/genetics
A simple multiplex PCR method for the concurrent detection of three CYP2C8 variants

Muthiah YD, Lee WL, Teh LK, Ong CE, Salleh MZ, Ismail R

Clin Chim Acta, 2004 Nov;349(1-2):191-8.
PMID: 15469873 DOI: 10.1016/j.cccn.2004.06.024

BACKGROUND: Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4.
METHOD: Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers.
RESULT: The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method.
CONCLUSION: We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.

Matched MeSH terms: DNA Primers
Fulltext A simple protocol to isolate DNA from Malaysian stork blood collected on FTA® cards

YEE, ELSIE Y. S., ZAINAL ZAHARI, AHMAD ISMAIL, YAP, C.K., TAN, S. G.

Buletin Persatuan Genetik Malaysia, 2009;14(1):20-22.
MyJurnal

The blood of the Painted Storks (Mycteria leucocephala) and the Milky Storks (M. cinerea) from Malaysia were collected
invasively from the breeding site. The blood was dropped on to FTA® cards and stored at room temperature. DNA was isolated from
the FTA® cards through a modification of the Wizard DNA Purification kit (Promega) procedure and PCR was performed with 11 pairs
of microsatellite primers of the American Wood Stork (M. americana). The collection of a drop of blood onto the card is superior to the
usual practice of collecting about five ml of blood into a vacuum tube as it causes fewer traumas to these sensitive birds. Moreover, this
collection procedure can be adopted for use in various wild animal species which are usually found in the remote areas of Malaysia as
the sample collection cards can be transported back to the laboratory at room temperature. Our procedure allows the typing of several
molecular genetic markers from just a drop of blood collected in the field and stored at room temperature alleviating the need for storage
in expensive deep freezers or liquid nitrogen tanks.

Matched MeSH terms: DNA Primers
A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

Ghaznavi-Rad E, Nor Shamsudin M, Sekawi Z, van Belkum A, Neela V

J Med Microbiol, 2010 Oct;59(Pt 10):1135-1139.
PMID: 20616192 DOI: 10.1099/jmm.0.021956-0

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Matched MeSH terms: DNA Primers/genetics
A thermoalkaliphilic lipase of Geobacillus sp. T1

Leow TC, Rahman RN, Basri M, Salleh AB

Extremophiles, 2007 May;11(3):527-35.
PMID: 17426920

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.

Matched MeSH terms: DNA Primers
A transposon-based genetic marker for conspecific identity within the Bactrocera dorsalis species complex

Zimowska GJ, Xavier N, Qadri M, Handler AM

Sci Rep, 2024 Jan 22;14(1):1924.
PMID: 38253542 DOI: 10.1038/s41598-023-51068-2

Here we describe a molecular approach to assess conspecific identity that relies on the comparison of an evolved mutated transposable element sequence and its genomic insertion site in individuals from closely related species. This was explored with the IFP2 piggyBac transposon, originally discovered in Trichoplusia ni as a 2472 bp functional element, that was subsequently found as mutated elements in seven species within the Bactrocera dorsalis species complex. In a B. dorsalis [Hendel] strain collected in Kahuku, Hawaii, a degenerate 2420 bp piggyBac sequence (pBacBd-Kah) having ~ 94.5% sequence identity to IFP2 was isolated, and it was reasoned that common species, or strains within species, should share the same evolved element and its precise genomic insertion site. To test this assumption, PCR using primers to pBacBd-Kah and adjacent genomic sequences was used to isolate and compare homologous sequences in strains of four sibling species within the complex. Three of these taxa, B. papayae, B. philippinensis, and B. invadens, were previously synonymized with B. dorsalis, and found to share nearly identical pBacBd-Kah homologous elements (> 99% nucleotide identity) within the identical insertion site consistent with conspecific species. The fourth species tested, B. carambolae, considered to be a closely related yet independent species sympatric with B. dorsalis, also shared the pBacBd-Kah sequence and insertion site in one strain from Suriname, while another divergent pBacBd-Kah derivative, closer in identity to IFP2, was found in individuals from French Guiana, Bangladesh and Malaysia. This data, along with the absence of pBacBd-Kah in distantly related Bactrocera, indicates that mutated descendants of piggyBac, as well as other invasive mobile elements, could be reliable genomic markers for common species identity.

Matched MeSH terms: DNA Primers
Fulltext Absence of nucleotide alteration in region of exon 34 of NOTCH1 and NOTCH2 receptor genes analysed in oral cancer samples: a preliminary observation

Nor Nasyitah Ismail, Khairani Idah Mokhtar

Archives of Orofacial Sciences, 2010;5(1):17-23.
MyJurnal

Oral cancer is one of the common cancer cases identified in the developing countries. Genetic mutation and overexpression of certain genes and proteins have been associated in the development of this cancer. Notch signalling pathway is normally involved in controlling the development process of vertebrates and invertebrates; however, deregulation of this pathway was found to be responsible in the formation of certain cancers including oral cancers. Activation of this pathway requires binding of the ligands to its receptors. Four NOTCH receptors (NOTCH 1, 2, 3 and 4) have been identified in mammals. Disruptions within these molecules might interfere with the normal functions of Notch signalling pathway. Hence, this study was conducted to detect mutations of NOTCH1 and NOTCH2 receptor genes which might be occurring in the oral cancer cases obtained from the local population. DNA extracted from fresh-frozen tissue biopsy of the tongue and buccal mucosa from 10 confirmed cases of oral cancer were subjected for polymerase chain reaction (PCR) amplification using the specific sets of primers. The PCR products were sent for sequencing before final results were analysed.
Due to time and cost limitation, only two out of four NOTCH receptor genes; NOTCH1 and NOTCH2, were used in this analysis. The results revealed absence of nucleotide changes for both NOTCH receptor genes amplified from these oral cancer samples. More samples and further analysis looking into other regions in these genes are required to conclude the involvement of NOTCH receptor genes mutation in causing oral cancer.

Matched MeSH terms: DNA Primers
Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and β-catenin and enhances invasion in nasopharyngeal carcinoma cells

Yip WK, Seow HF

Cancer Lett, 2012 May 28;318(2):162-72.
PMID: 22182447 DOI: 10.1016/j.canlet.2011.12.018

Dysregulation of E-cadherin and β-catenin function in cell-cell adhesion is common in nasopharyngeal carcinoma (NPC) and correlates with metastatic disease. In this study, we examined the role of EGF-activated phosphatidylinositol 3-kinase (PI3K)-Akt signaling in E-cadherin and β-catenin regulation. We found that reduced membranous E-cadherin and β-catenin expression was positively correlated with Akt phosphorylation in NPC tissues. EGF treatment disrupted cell-cell adhesion and resulted in mesenchymal morphological features in NPC cell lines (TW01, TW04, and TW06). Western blot analysis showed that the E-cadherin protein level was partially reduced in TW04 cells only and the β-catenin levels were not considerably affected upon EGF treatment. In contrast, quantitative real-time RT-PCR showed that the E-cadherin, but not β-catenin, mRNA levels were markedly reduced by EGF in all cell lines. Immunofluorescent staining revealed that E-cadherin and β-catenin appeared to be markedly reduced on the cell surface and more localized in the cytoplasm. Inhibition of PI3K by LY294002 did not abolish the EGF-induced downregulation of E-cadherin protein or mRNA in TW04 cells but moderately increased the β-catenin protein level in TW01 cells and mRNA level in TW06 cells. However, LY294002 substantially restored or increased cell surface E-cadherin and β-catenin in all EGF-treated cell lines, in concordance with the inhibition of cell morphological changes. Moreover, LY294002 significantly blocked EGF-driven cell invasion, correlating with the elevation of membranous E-cadherin and β-catenin levels. In conclusion, EGF-induced epithelial-to-mesenchymal transition may not be only dependent on downregulation of E-cadherin protein/mRNA but also on mislocalization of E-cadherin and β-catenin. The mechanisms involved may be related, at least in part, to the PI3K-Akt pathway.

Matched MeSH terms: DNA Primers
Alterations of the p14ARF-p53-MDM2 pathway in oral squamous cell carcinoma: MDM2 overexpression is a common event

Lim KP, Sharifah H, Lau SH, Teo SH, Cheong SC

Oncol Rep, 2005 Oct;14(4):963-8.
PMID: 16142358 DOI: 10.3892/or.14.4.963

The majority of global incidences of oral cancer occur in Asia, and the aetiology of oral cancer is different in Asia as it is in the West. However, whereas there is a growing understanding of the molecular mechanisms of oral cancer progression in the West, there is little progress in this understanding in Asia. In particular, the role of the p53 pathway in modulating cancer progression in Asian oral cancer remains unclear. In this study, we micro-dissected and analysed 20 well-differentiated oral squamous cell carcinoma specimens for alterations in the p53 pathway. We found that 6/20 samples contained mutations in the p53 gene which occurred in three hotspots, at codon 203, 218 and 296. Furthermore, 6/20 samples had a homozygous deletion of p14ARF, but notably p14ARF deletion and p53 mutation events were often independent and mutually exclusive. Strikingly, MDM2 was upregulated in 20/20 samples, but not in 3/3 normal tissue specimens. Taken together, these data suggest that inactivation of the p53 pathway is a frequent event in oral squamous cell carcinoma, which occurs by an aberration in one of a number of players in the p53 pathway.

Matched MeSH terms: DNA Primers/chemistry
An assay for selection of sera with circulating Toxoplasma gondii antigens

Emelia O, Zeehaida M, Sulaiman O, Rohela M, Saadatnia G, Yeng C, et al.

J Immunoassay Immunochem, 2010;31(1):79-91.
PMID: 20391020 DOI: 10.1080/15321810903405134

We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.

Matched MeSH terms: DNA Primers
Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling

Learn-Han L, Yoke-Kqueen C, Salleh NA, Sukardi S, Jiun-Horng S, Chai-Hoon K, et al.

Antonie Van Leeuwenhoek, 2008 Oct;94(3):377-87.
PMID: 18548329 DOI: 10.1007/s10482-008-9254-y

Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.

Matched MeSH terms: DNA Primers/genetics

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