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Appl. Environ. Microbiol. 72, 2539-2546. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. 2006

Mohan Nair, M.K. and Venkitanarayanan, K.S.

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconstituted infant formula. (3464)

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Exp. Parasitol. 112, 63-65. Babesia canis vogeli: A novel PCR for its detection in dogs in Australia. 2006

Martin, A.R., Dunstan, R.H., Roberts, T.K., and Brown, G.K.

Notes: GoTaq® DNA Polymerase was used in PCR to test dog blood for the presence of Babesia canis. Genomic DNA isolated from dog blood was analyzed with primers to the variable 5’ region of the Babesia canis 18S rRNA gene. PCR was performed in 50µl reactions containing 1.25 units of GoTaq® DNA Polymerase and 10µl of GoTaq® Reaction Buffer. (3367)

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Appl. Environ. Microbiol. 72, 5463–5468. Comparative, collaborative, and on-site validation of a TaqMan® PCR method as a tool for certified production of fresh, campylobacter-free chickens. 2006

Krause, M., Josefsen, M.H., Lund, M., Jacobsen, N.R., Brorsen, L., Moos, M., Stockmarr, A. and Hoorfar, J.

Notes: To test if a real-time PCR assay would be able to detect Campylobacter spp. in various chicken samples, several laboratories were involved in a collaborative trial. Each lab was given 18 1ml samples, including 6 chicken neck skin samples, 6 shoe cover fecal samples and 6 cloacal swab samples. DNA was extracted from these samples using the MagneSil® KF, Genomic System, first tested using 20µl of paramagnetic particles (PMPs) and then using 75µl PMPs. Five microliters of purified DNA was used in real-time PCR analysis. (3558)

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J. Clin. Microbiol. 44, 3878–82. Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. 2006

Le Guyader, F.S., Bon, F., DeMedici, D., Parnaudeau, S., Bertone, A., Crudeli, S., Doyle, A., Zidane, M., Suffredini, E., Kohli, E., Maddalo, F., Monini, M., Gallay, A., Pommepuy, M., Pothier, P. and Ruggeri, F.M.

Notes: The authors linked outbreaks of acute gastroenteritis in Italy and France with consumption of oysters contaminated with Norovirus. In Italy Norovirus RNA was detected in fecal samples from infected individuals using the Access RT-PCR System and primers for the Norovirus polymerase region. (3792)

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J. Clin. Microbiol. 44, 3285-3291. Rapid and sensitive detection of single Cryptosporidium oocysts form archived glass slides. 2006

Sunnotel, O., Snelling, W.J., Xiao, L., Moule, K., Moore, J.E., Millar, B.C., Dooley, J.S.G. and Lowery, C.J.

Notes: These researchers used laser-capture microscopy followed by real-time PCR to detect and identify Cryptosporidium oocysts in stained fecal smears and water samples on glass slides. After microdissection of single oocysts or groups of oocysts from the stained slides, DNA was extracted and real-time PCR performed using primers specific for the cryptosporidial 18s rRNA gene. To confirm primer specificity and the identity of the real-time PCR products, the amplimers were recovered from the LightCycler® capillaries at the end of each each real-time experiment. Products were then separated on agarose gels and purified using the Wizard® SV Gel and PCR System prior to sequencing using a BigDye® terminator cycle sequencing kit from Applied Biosystems. (3532)

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Clin. Chem. 52, 2250–2257. Sensitive detection of KIT D816V in patients with mastocytosis. 2006

Tan, A., Westerman, D., McArthur, G.A., Lynch, K., Waring, P. and Dobrovic, A.

Notes: The authors wanted to develop a more sensitive assay to detect codon 816 pathogenic variations in people diagnosed with systemic mastocytosis. The Wizard® Genomic DNA Purification Kit was used to isolate DNA from peripheral blood and bone marrow aspirate samples. To extract DNA from 2–5 micron, paraffin-embedded samples of bone marrow trephine, skin, spleen or liver, the tissues were digested with Proteinase K for four days at 56°C prior to DNA purification using the Magnesil® Genomic Fixed Tissue System. The isolated DNA was subjected to two assays: enriched sequencing of mutant alleles (ESMA) after BsmAI restriction enzyme digestion, and allele-specific competitive blocker PCR (ACB-PCR). (3575)

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Anal. Bioanal. Chem. 385, 1045-1054. Effective PCR detection of animal species in highly processed animal byproducts and compound feeds. 2006

Fumière, O., Dubois, M., Baeten, V., von Holst, C. and Berben, G.

Notes: The authors developed a PCR method to detect the presence of meat and bone meal (MBM) in animal feed even if the MBM had been heat treated, and discern whether the animal component is bovine or porcine in origin. The genomic DNA from 100mg of various feedstuffs with known and unknown amounts of MBM, fishmeal or poultry feed or a combination of these compounds was isolated using the Wizard® Magnetic DNA Purification System for Food with the KingFisher System. Real-time PCR was performed using 5μl of extracted DNA. (3750)

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Pesticide Biochem. Physiol. 84, 236-242. Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster. 2006

Hardstone, M.C., Baker, S.A., Gao, J., Ewer, J., and Scott, J.G.

Notes: Researchers used the GoTaq® DNA Polymerase in PCR screens for excisions around a CYP6d4 gene in the HA-1829 strain of Drosophila. PCR was performed in a 20μl reaction volume using 0.4 Units of GoTaq® DNA Polymerase, 2.75mM MgCl2 and 1μl of DNA (equivalent to the DNA in approximately 1/5 to 1/10 of a fly). (3363)

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Appl. Environ. Microbiol. 72, 6070-6078. An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae. 2006

Taylor, T.V., Mitchell, T.K. and Daub, M.E.

Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard® SV Gel and PCR Clean-Up System, and subcloned into the pGEM®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

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J. Clin. Microbiol. 44, 2750–2759. Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. 2006

Zucol, F., Ammann, R.A., Berger, C., Aebi, C., Altwegg, M., Niggli, F.K., and Nadal, D.

Notes: A panel of 11 Gram-negative and 11 Gram-positive bacterial species was used to develop a real-time PCR detection method. Initially DNA was purified from 1ml of various dilutions of bacteria resuspended in a saline solution using either the QIAamp DNA blood mini kit or the Wizard® SV Genomic DNA Purification System. The results suggested that the Wizard® SV Genomic DNA Purification System extraction protocol was superior in disrupting the bacterial cell wall (especially of Gram-positive bacteria), to allow release of bacterial DNA. Using DNA purified with the Wizard® SV Genomic DNA Purification System, detection of S. aureus and E. coli at concentrations as low as 101 CFU per PCR was achieved. The Wizard® SV Genomic DNA Purification System purification protocol provided in eNotes online (www.promega.com/enotes/applications/ap0051_tabs.htm) was used with the following modifications: The bacterial pellet was resuspended in 400µl of enzymatic lysis solution (47mM EDTA, 25mg/ml lysozyme, 20µg/ml lysostaphin) and incubated for 2 hours at 37°C. Next, 19.2mg/ml proteinase K was added (final concentration 0.4mg/ml), and the mixture was incubated for 1 hour at 55°C. Finally, the Nuclei Lysis Solution and RNase Solution were added, mixed and incubated for 10 minutes at 80°C. For real-time PCR analysis, 2µl of genomic DNA was used. (3676)

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Plant Sci. 170, 705-14. Expression profile analysis and biochemical properties of the peptide methionine sulfoxide reductase A (PMSRA) gene family in Arabidopsis. 2006

Romero, H.M., Pell, E.J. and Tien, M.

Notes: PMSRA expression was examined in 4-week old plants exposed to 10μM methyl viologen, 100μM cercosporin, photo-oxidative stress or ozone. Samples were ground in liquid nitrogen and total RNA isolated. Total RNA (2.5μg) was reverse transcribed into cDNA with random primers d(N)10, then amplified using gene-specific primers for PMSRA1—PMSRA5 and an antibody-mediated hot start containing a mixture of GoTaq® DNA Polymerase and Taq antibody (BD Biosciences, Mountain View CA). In a total volume of 25μl, the RT-PCR reaction mixture contained 2.5 units of GoTaq® DNA Polymerase, 10mM Tris–HCl (pH 8.5), 60mM KCl, 2.4mM MgCl2 and 300μM of each dNTP. For each RT-PCR, a plant 18S universal internal standard (Ambion, Austin TX) was included as a loading control. Amplification reaction conditions were as follows: 27 cycles at 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute. For each analysis, three rounds of RT-PCR were conducted with three independently isolated total RNA samples. Twenty microliters from each PCR were fractionated by 1.5% (w/v) agarose gel in Tris–borate EDTA buffer and stained with 0.5% (w/v) ethidium bromide. (3370)

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Protein Expr. Purif. 47, 562–570. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. 2006

Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.

Notes: In this study, the Flexi® Vector Systems was compared with the Gateway® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway® vector and the equivalent Flexi® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi® Vector system was shorter at 6–8 days compared to 12 days for the Gateway® system. They also found the Flexi® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil® PCR Clean-Up System or Wizard® SV 96 PCR Clean-Up, ligated into the Flexi® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi® Vectors and transfer of cDNA inserts between different Gateway® vectors and found similar performance in the two systems. For the Flexi® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi® Vectors. (3533)

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J. Exp. Bot. 57, 3737–46. Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. 2006

Yao, Q., Cong, L., Chang, J.L., Li, K.X., Yang, G.X. and He, G.Y.

Notes: The authors produced transgenic wheat plants with low-copy insertion of 1Ax1 and bar genes without the concomitant insertion of vector sequences using particle bombardment. Tissue-specific expression of the 1Ax1 gene, which has an endosperm-specific promoter, was examined using RT-PCR in 21 transgenic wheat lines. Total RNA was isolated from leaf, endosperm, root and inflorescence tissues, then amplified using the Access RT-PCR System. (3793)

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J. Med. Microbiol. 55, 273–277. Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes. 2006

Nakayama, A., Okayama, A., Hashida, M., Yamamoto, Y., Takebe, H., Ohnaka, T., Tanaka, T. and Imai, S.

Notes: In this study, a real-time PCR assay coupled with a DNA extraction method were used to detect staphylococcal enterotoxin (SE)-encoding genes in milk, a source of staphylococcal food poisoning. Pasteurized milk prepared with known concentrations of Staphylococcus aureus was used to generate a standard curve; experimental samples were from a staphylococcal food-poisoning outbreak that occurred in Japan in June 2000. PCR inhibition was overcome by using the following DNA purification method: a sample of milk (100µl) was added to an equal volume of 0.2M sodium hydroxide and incubated at 37°C for 20 minutes. The alkaline-treated sample was neutralized using 10µl of 3M sodium acetate (pH 5.4), extracted with 1ml of petroleum ether, and centrifuged at 13,000 × g for 10 minutes at 25°C. The aqueous phase was transferred to a fresh tube and bacterial DNA was purified from the aqueous solution using the Wizard® SV Genomic DNA Purification System. (3677)

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Clin. Chem. 52, 1070-1079. Quantitative reverse transcription-pcr assay for detection of mRNA encoding full-length human tissue kallikrein 7: Prognostic relevance of KLK7 mRNA expression in breast cancer. 2006

Holzscheiter, L., Biermann, J.C., Kotzsch, M., Prezas P., Farthmann, J., Baretton, G., Luther, T., Tjan-Heijnen, V.C.G., Taliere, M., Schmitt, M., Sweep, F.C.G.J., Span, P.N. and Magdolen, V.

Notes: Few reliable prognostic markers have been developed for breast cancer. Human tissue kallikreins (hKs) are serine proteases with diverse functions. Fifteen human tissue kallikrein genes (KLK) have been identified. Several human tissue kallikreins have been associated with malignancies, such as hK3 (prostate-specific antigen) and prostate cancer. hK7 has been linked to a significantly poorer prognosis for ovarian and breast cancer, indicating that it may serve as a marker for these diseases. However, previous analyses of KLK7 expression were non-selective studies of all KLK7 mRNA forms. The authors developed a quantitative reverse-transcription-PCR (QPCR) assay, using the Reverse Transcription System on RNA, then developed a highly sensitive QPCR assay, to determine whether a more specific analysis of full-length KLK7 mRNA might lead to similar results as those from previous studies on the multiple KLK7 mRNA forms, with the ultimate goal of determining whether KLK7 could serve as a marker for breast cancer. (3615)

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J. Neurosci. 26, 3299-3308. Human astrocytes are resistant to Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. 2006

Song, J.H., Bellail, A., Tse, M.C.L., Yong, V.W. and Hao, C.

Notes: Total RNA was extracted from human astrocytes and control A549 cells. First strand cDNA was synthesized from 3μg of total RNA using random hexamers. PCR was performed on the cDNA samples using primers for DR4, DR5, and GAPDH with GoTaq® Green Master Mix. The PCR was performed with an initial denaturation at 94°C for 2 minutes, followed by cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 45 seconds at 72°C. The PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. (3372)

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Nucl. Acids Res. 34, 584-592. Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence. 2006

Nehdi, A. and Perreault, J.P.

Notes: To characterize sequence variability of the catalytic center of the hepatitis delta virus (HDV) ribozyme, which includes a self-cleaving motif, the authors developed an in vitro selection process that allowed them to select self-cleaving sequence variants from a pool of HDV ribozymes. The selection process started with a library of DNA oligonucleotides corresponding to the HDV ribozyme sequence that had been randomized at a 25-nucleotide sequence. The oligos contained known sequences at the 5´ and 3´ ends to allow amplification. The library was amplified by PCR, then transcribed. The resulting RNA was separated by polyacylamide gel electrophoresis, and the self-cleaved transcripts were excised, purified and reverse transcribed. A 3´ extension using terminal deoxynucleotidyl transferase added a known sequence to the 3´ end so that the resulting cDNA could by amplified by PCR. Thus another cycle of selection could begin. The PCR products generated at the start of the process and at each cycle were cloned into the pGEM®-T Vector. (3467)

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Vet. Parasitol. 135, 99-104. Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification. 2006

Theodoropoulos, G., Gazouli, M., Ikonomopoulos, J.A., Kantzoura, V., and Kominakis, A.

Notes: Researchers used GoTaq® DNA Polymerase to test sheep and goat blood samples for the presence of Babesia DNA. Primers were designed around the 18S rRNA sequence of Babesia sp. PCR was performed in a 50µl reaction volume using 1 unit of GoTaq® DNA Polymerase. Ten microliters of each amplification reaction were loaded on gels and subjected to electrophoresis. (3380)

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FEBS Lett. 579, 832-8. Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways. 2006

Takami, M., Cho, E.S., Lee, S.Y., Kamijo, R. and Yim, M.

Notes: Mouse bone marrow cells and calvarial osteoblasts were cocultured for 6 days with or without 50 μM of IBMX. Total RNA was then isolated from the cells and cDNA templates prepared. cDNAs were subjected to PCR amplification with GoTaq® DNA Polymerase. Primers for mouse PDE4s, TRANCE, CTR, cathepsin K and GAPDH genes were used in this study. The PCR program was as follows: 32 (all mouse PDE4s, TRANCE, CTR, and cathepsin K) or 28 (GAPDH) cycles, after an initial denaturation step at 94°C for 3 minutes, then denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, with a final extension at 72°C for 10 minutes. (3356)

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Hum. Mol. Genet. 15, 999–1013. An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements. 2006

Disset, A., Bourgeois, C.F., Benmalek, N., Claustres, M., Stevenin, J. and Tuffery-Giraud, S.

Notes: To construct dystrophin minigenes, genomic DNA containing a mutation in dystrophin was amplified for exons 30, 31 and 32. The three PCR fragments were combined and amplified into one product. This overlap-extension PCR generated two minigenes which were then cloned into the pGEM®-T Vector and sequenced. After EcoR I digestion, the minigenes were ligated into the pSI Mammalian Expression Vector and transiently transfected into C2C12 cells for expression analysis. (3499)

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Nucl. Acids Res. 34, 2109–2116. The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription. 2006

Roberti, M., Bruni, F., Polosa, P.L., Gadaleta, M.N. and Cantatore, P.

Notes: To examine if the depletion of Drosophila transcription termination factor (DmTTF) after RNAi treatment could reduce the gene copy number, genomic DNA was isolated from RNAi-treated and untreated Drosophila embryonic D.Mel-2 cells using the Wizard® Genomic DNA Purification Kit. The mitochondrial ND3 gene and the nuclear H2B histone gene were used as probes for the Xho I-digested, Southern-blotted genomic DNA to compare the treatment groups. The Wizard® SV Gel and PCR Clean-Up System was used to clean up the PCR-amplified probes. (3418)

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Nucl. Acids Res. 34, e67. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. 2006

Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E. and Sverdlov, E.

Notes: The authors selected repetitive elements in the human genome using a novel technique: GREM. T4 DNA Ligase was used to ligate adapters to digested genomic DNA prior to PCR, and exonuclease III was used to generate the necessary 5´ termini. After the final amplification, the PCR products were cloned into the pGEM®-T Vector, then sequenced. (3550)

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Vet. Immunol. Immunopathol. 110, 279-92. Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells. 2006

Denyer, M.S., Wileman, T.E., Stirling, C.M.A., Zuber, B., and Takamatsu, H.

Notes: In this study, GoTaq® DNA Polymerase was used in two-step RT-PCR. The ImProm-II™ Reverse Transcription System was first used to produce cDNA using an oligo d(T)15 primer. PCR was then performed using GoTaq® DNA Polymerase. Each reaction contained 2μl cDNA, 10μl GoTaq® Reaction Buffer, 1μl dNTP (10mM), 0.2μl GoTaq® DNA Polymerase, 1μl each primer (10pmol) and 34.8μl nuclease-free water. PCR was performed at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds for 35 cycles, and 72°C for 10 minutes.PCR products were visualized by agarose gel electrophoresis containing ethidium bromide and then sequenced.

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Clin. Chem. 52, 634-642. Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis. 2006

Mitsuhashi, M., Tomozawa, S., Endo, K. and Shinagawa, A.

Notes: The authors developed a method to reverse transcribe poly(A)+ RNA from leukocytes without using oligo(dT) immobilized on a 96-well plate. Four RNA targets, as well as a synthetic control RNA, were reverse transcribed using MMLV Reverse Transcriptase (1X reverse transcription buffer [50mM KCl, 10mM Tris-HCl (pH 8.3), 5.5mM MgCl2, 1nL/µL Tween 20], 1.25 mM each dNTP and 4 units of Recombinant RNasin® Ribonuclease Inhibitor), then quantitated by real-time quantitative PCR. The authors varied the concentration of MMLV Reverse Transcriptase, incubation time, and primer/template ratio] to obtain the maximum yield. Small quantities of MMLV Reverse Transcriptase were sufficient to reverse transcribe short synthetic RNA and abundant RNAs, but approximately 100 units was required for other RNAs. The synthetic control RNA was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. (3456)

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J. Biol. Chem. 281, 13199-13208. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity. 2006

Rosenkilde, M.M., Benned-Jensen, T., Andersen, H., Holst, P.J., Kledal, T.N., Luttichau, H.R., Larsen, J.K., Christensen, J.P. and Schwartz, T.W.

Notes: The expression level of the seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) was measured in peripheral blood mononuclear cells. Total RNA was isolated from T-lymphocytes, B-lymphocytes, monocytes and NK cells, and reverse transcribed using the ImProm-II™ Reverse Transcription System. The resulting cDNA was quantitated using real-time PCR. (3449)

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