Species Differentiation Of Fish Samples By Restriction Fragment Length Polymorphism Analysis Of Cytochrome B Gene

Metode pengukuran polimorfisme fragmen hasil pemotongan produkreaksi polimorfik berantai oleh enzim restriksi spesifik (polymerase chainreaction-restriction fragment length polymorphism, RFLP-PCR) telah digunakanuntuk membedakan beberapa jenis ikan mentah. Situs cytochrome b mitokondria,yang diamplifikasi oleh primer universal, dipotong menggunakan empat enzimrestriksi (Bfa I, Hinf I, Msp I, Mbo II) sehingga dapat dianalisa fragment-fragmentpendeknya. Hasil yang diperolah dari pemotongan oleh enzim restriksi tersebutternyata dapat digunakan untuk membedakan tiap jenis ikan sampel. Hasilpenelitian ini menunjukkan bahwa PCR dan RFLP-PCR merupakan metode yangsensitif dan dapat dilakukan dalam waktu singkat untuk membedakan berbagaijenis ikan mentah

PCR-RFLP Using BseDI Enzyme for Pork Authentication in Sausage and Nugget Products

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BseDI restriction enzyme had been applied for identifying the presence of pork in processed meat (beef sausage and chicken nugget) including before and after frying. Pork sample in various levels (1%, 3%, 5%, 10%, and 25 %) was prepared in a mixture with beef and chicken meats and processed for sausage and nugget. The primers CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b (cyt b) gene and PCR successfully amplified fragments of 359 bp. To distinguish existence of porcine species, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed pig mitochondrial DNA was cut into 131 and 228 bp fragments. The PCR-RFLP species identification assay yielded excellent results for identification of porcine species. It is a potentially reliable technique for pork detection in animal food processed products for Halal authentication

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the α1, α2, β, γ1, γ2/θ, κ, ɛ, ζ, and ι intimin variants

Genetic diversity among viruses associated with sugarcane mosaic disease in Tucumán, Argentina

Sugarcane leaves with mosaic symptoms were collected in 2006--07 in Tucumán (Argentina) and analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) and sequencing of a fragment of the Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) coat protein (CP) genes. SCMV was detected in 96.6% of samples, with 41% showing the RFLP profile consistent with strain E. The remaining samples produced eight different profiles that did not match other known strains. SCMV distribution seemed to be more related to sugarcane genotype than to geographical origin, and sequence analyses of CP genes showed a greater genetic diversity compared with other studies. SrMV was detected in 63.2% of samples and most of these were also infected by SCMV, indicating that, unlike other countries and other Argentinean provinces, where high levels of co-infection are infrequent, co-existence is common in Tucumán. RFLP analysis showed the presence of SrMV strains M (68%) and I (14%), while co-infection between M and H strains was present in 18% of samples. Other SCMV subgroup members and the Sugarcane streak mosaic virus (SCSMV) were not detected. Our results also showed that sequencing is currently the only reliable method to assess SCMV and SrMV genetic diversity, because RT-PCR-RFLP may not be sufficiently discriminating.Fil: Perera, María Francisca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Filippone, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Ramallo, C. J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Cuenya, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Garcia, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Ploper, Leonardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; Argentin

Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular

Infection of Gammarus duebeni populations by two vertically transmitted microsporidia; parasite detection and discrimination by PCR–RFLP

We screened a population of the brackish water crustacean Gammarus duebeni from the Isle of Cumbrae for the presence of vertically transmitted microsporidia. We compared 2 screening techniques; light microscopy and PCR-based detection using generic 16S rDNA microsporidian primers. Fifty percent of females from this population tested positive for vertically transmitted microsporidia. The PCR screen was 100% efficient in comparison with existing LM based screening. In addition, the PCR screen produced bands of 2 sizes suggesting that more than 1 species of microsporidian was present. Sequencing revealed 2 distinct species of vertically transmitted microsporidia; 33% of females were infected with the feminizer Nosema granulosis and 17% were infected with a new species which we provisionally designate Microsporidium sp. On the basis of sequence information, we developed a discriminatory PCR–RFLP test based on MspI and HaeIII digests. This screen allows rapid detection and discrimination of vertically transmitted microsporidia in natural field populations. We applied the PCR–RFLP screen to a second G. duebeni population from the Isle of Man. This population also hosted these 2 parasite species. In total 45% of females harboured N. granulosis and 10% harboured Microsporidium sp. No dual-infected individuals were found in either population. The occurrence of 2 vertically transmitted parasites within a population has implications for our understanding of parasite–host relationships in the field and we discuss factors affecting the dynamics of parasite–parasite competition and coexistence

Validation of a Polymerase Chain Reaction technique for Kidd blood group genotyping

The Kidd blood group antigens, Jkª and Jkᵇ , are two of the main surface markers which are found on the membrane of red blood cells. The determination of whether a donor or a recipient has the Jkª and/or the Jkᵇ antigens is crucially important to have a successful transfusion without the development of adverse incompatibility-related reactions. In Malta, routine serological-based tests are applied with the purpose of differentiating between homozygous and heterozygous states for the Jk antigens respectively. Although these tests are highly specific and sensitive, there are particular clinical scenarios where haemagglutination assays are not suitable for determining the individual’s Kidd blood group status. Additionally, the alternative genotyping procedure has never been applied in Malta within the context of blood grouping. The current study was therefore carried out to determine whether a molecular-based technique such as Polymerase Chain Reaction – Restriction Fragment Length Polymorphism analysis (PCR-RFLP) is a suitable alternative procedure for distinguishing amongst the three different Kidd phenotypes. After extracting deoxyribonucleic acid (DNA) from 50 blood samples obtained from serologically-tested healthy blood donors who expressed at least one of the Kidd antigens, PCR-RFLP analyses were carried out. The results of the latter were then compared with those previously obtained with haemagglutination and a complete match was observed between the two. Therefore, this PCR-RFLP method was confirmed as a suitable alternative laboratory technique that can be used to determine efficiently the Kidd blood group of both donors and recipients, in an accurate manner without subjectivity as encountered in the case of haemagglutination. This research further facilitates the introduction of molecular-based techniques in molecular blood transfusion.peer-reviewe