Bp Polymerase Chain Reaction Product Research Articles

A 3D-printed oscillatory polymerase chain reaction system using a single heater

Nucleic acid amplification is a process that arises in all organisms. Polymerase chain reaction (PCR) is a standard laboratory nucleic acids amplification technology in vitro widely used in medical detection and biometric-related research. This device represents the first demonstration of 3D-printed oscillatory thermocycling within a portable PCR device. This study uses a single Peltier element to create the three temperature regions required for PCR. The overall control system uses an Arduino microcontroller with the advantages of easy development, abundant resources, and easy access to device software design. The sample-driving system integrated with the 3D printed object and the laser-cut acrylic component to achieve the linear reciprocating motion of the sample is conducive to the realisation of the portable design of the device. After system optimisation, the reaction tube moves from the denaturation zone to the aluminum block for cooling, and it moves to the groove of the denaturation zone from the annealing zone to accelerate the sample temperature rise. The time duration reduces from 180 s to 85 s to complete one thermal cycle. Combining the 5 mm instead of 3 mm thickness of the acrylic material and the configuration of the acrylic end exposed in the air creates a more significant temperature difference from 25 °C to 40 °C. Using this oscillatory PCR device, we successfully amplified a 219 bp PCR product of turmeric DNA after 30 thermal cycles within 50 min. The developed PCR device will cost less than $300 USD in our work. Compared with previous works, the cost of this research device is relatively low, and the total volume of the device is quite small. This work should be interesting to persons involved in diagnosing the infectious diseases of viruses and bacteria and identifying the fake healthy food in the low-cost bioreaction chambers in fields and remote areas.

A comparative study of serology and PCR for the diagnosis of brucellosis in goats

Brucellosis is an economically important infectious disease of livestock causing abortions, infertility, delayed oestrus, interrupted lactation, increased condemnation and loss of milk production besides its zoonotic nature. The present study was conducted to compare serological assays and polymerase chain reaction (PCR) for the diagnosis of caprine brucellosis. A total of 301 whole blood samples to extract DNA for PCR and serology were collected from goats maintained at various organized herds, panjarapoles, slaughter house, local meat markets, etc. in South Gujarat region of India. Out of 301 serum samples tested, 7 samples (2.33%) were positive by all the three serological tests, viz. rose bengal plate test (RBPT), indirect ELISA (iELISA) and immunochromatographic assay (ICA). Among 301 DNA samples, genus specific PCR detected DNA of Brucella spp. in 11 samples by targeting BCSP 31 and IS-711 genes to get 223 bp and 350 bp PCR products on agarose gel electrophoresis. None of the seven serologically positive samples showed Brucella genus-specific DNA amplification by PCR and similarly all PCR positive samples were negative on serology.

Specific PCR primers for the identification of Salmonella enterica serovar enteritidis in chicken-related samples

In this study, a designed pair of PCR primers, SefB127L-SefB661R, based on the sefb gene (accession number L11009) sequences was used in polymerase chain reaction (PCR) for rapid evaluation of Salmonella enterica serovar Enteritidis in chicken-related samples. The specificity of this method was checked with 85 Salmonella strains and 17 non-Salmonella strains. The results showed that only 24 isolates of S. enterica serovar Enteritidis exhibited 535 bp PCR product. The detection limit of this PCR method were evaluated using 40 spiked samples under enrichment protocols. The data revealed that microbial extract from as few as 101 target cells per gram of the sample culture was required for this assay. Before PCR amplification, pre-culture and the cell lysates, rather than the DNA extracts, were used directly for all tested samples. To verify the usefulness of this PCR process for S. enterica serovar Enteritidis examination, 285 endogenous samples including chicken meats, eggs and swabs of chicken-related samples and coop's facilities were tested and compared with that obtained by conventional BAM (Bacteriological Analytical Manual) method. About 1% (three in 285) of the S. enterica serovar Enteritidis samples was contaminated, approximately the same as that obtained from BAM method.

The determination of meat species by PCR-RFLP method using mitochondrial ND4 gene in pastırma, a traditional dry cured meat product

Pastirma is a high value, traditional, dry cured, and edible coated meat product. For economic and sociocultural reasons, it is important to know which meat species are used in pastirma. The present study aims to carry out the determination of the meat species in pastirma. A total of 144 pastirma samples were collected from different stores. After genomic DNA isolation, the total DNAs obtained were subjected to polymerase chain reaction (PCR) using specially designed novel primers amplifying the mitochondrial ND4 (MTND4) gene region, specific for cattle (Bos taurus), water buffalo (Bubalus bubalis), horse (Equus caballus), and donkey (Equus asinus) species. For the identification of the meat species, gel purified 952 bp PCR products were digested with fast digest SaqAI restriction enzymes selected based on preliminary in silico analyses. The results of the present study revealed that all of the pastirma samples were made from cattle meat. Cross-reactions and false-positive identifications are serious problems in routine determination of the meat species in food control laboratories. It is important to perform fast, inexpensive determination with high sensitivity and specificity for the identification of meat species. This study has shown that meat species can be detected with high specificity in products such as pastirma, which uses large pieces of meat cuts.

Sequencing and Phylogenetic Analysis of the Salmonella Enterotoxin (stn) Gene of Salmonella spp. Isolated from Egyptian Broiler Breeder Chickens Farms

Salmonella is a member of Enterobacteriaceae family that found to be pathogenic to domestic and wild animals and humans. Salmonellae were isolated from three distinct governorates, Giza, Monofia and Qaluobia from broiler breeder chicken farms. Molecular characterization of the Salmonella isolates using polymerase chain reaction (PCR) assay as well as Sequencing and phylogenetic analysis of PCR products were conducted to distinguish the collected Salmonellae species. The nucleotide sequence of 617 bp PCR products representing the amplified fragment of stn gene of seven isolates of Salmonella enteritidis has been sequenced. Furthermore, the nucleotide sequence was submitted to the gene bank. The obtained sequences were blasted with the highly similar sequences and the multiple sequence alignments were conducted. Neighbor-joining tree was constructed for the Egyptian Salmonella isolates against 30 Salmonella spp. from the Gene bank database representing maximum similarity with stn gene when subjected to multiple sequence alignment, and the phylogenetic tree was constructed based on the comparative analysis of related sequences at the nucleotide level.

Genetic diversity of Prolactin gene in Japanese quail (Coturnix Coturnix Japonica)

This study was carried out to investigate the genetic diversity of prolactin gene (PRL gene) in Japanese quails using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) marker. One hundred and thirty nine quails were sampled for PRL loci analysis. DNA was extracted from the samples. Polymerase chain reaction (PCR) and electrophoresis was used to characterize a 24 base pair (bp) insertion/deletion in a 358 bp PCR product. Test for Hardy-Weinberg Equilibrium were carried out. From the results, two alleles A (0.63) and B (0.37) and three genotypes AA, AB and BB were observed from this locus. Results of the study showed that genetic variability exists in the study population of Japanese quails in Nigeria and its segregation could be assessed for reproductive capacity

Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

MOLECULAR IDENTIFICATION OF SARCOPTES SCABIEI VAR. CUNICULI FROM SURABAYA AND MALANG REGIONS OF EAST JAVA

Scabies is a zoonotic skin disease caused by Sarcoptes scabiei mites. As an emerging/re-emerging parasitic disease, scabies represents a significant global threat to both human and animal health. Numerous cases of scabies in Indonesia have been reported, which support research on the prevalence of S. scabiei. However, most such studies have involved conventional morphological studies, with limited molecular diagnostic studies. The purpose of the present study was the genetic characterization of S. scabiei var. cuniculi in domestic rabbits to generate baseline genotypic data. S. scabiei var. cuniculi was isolated and identified from scabies-infected rabbits from the Surabaya and Malang regions of East Java. Molecular identification was performed using Polymerase Chain Reaction (PCR) using specific primers targeting the COX1 gene. We performed COX1 PCR using rabbit isolates of S. scabiei from Indonesia. To the best of our knowledge, no such study had been reported previously. This study was performed in the Laboratory of Veterinary Parasitology, Faculty of Veterinary Medicine and the Tropical Disease Diagnostic Center Laboratory, Universitas Airlangga. The results with agarose gel electrophoresis revealed a 289 bp PCR product amplified from the DNA of S. scabiei isolates from both Surabaya and Malang in accordance with the expected COX1 amplicon size, that indicated a single band 289 bp in length, demonstrating specific detection of S. scabiei var. cuniculi from Surabaya and Malang using COX1 primers. The results were consistent with the calculated amplicon size based on primer positions within the COX1 locus, with the forward primer spanning nucleotides 61–94, and the reverse primer spanning nucleotides 331–350 ( 350 − 61 = 289 bp). PCR genotyping of the isolates yielded an identical nucleotide length of 289 bp. Further studies are required to sequence the amplified fragments for homology assessment.

PARASITOLOGICAL AND MOLECULAR STUDIES ON TRYPANOSOMA EVANSI OF CAMELS IN EGYPT

This study was conducted on a total of 187 one humped camels (Camelus dromedarius). Blood samples were collected from 40 camels suspected forTrypanosoma infection from farms at Giza Pyramids area. In addition, 147 samples were collected from apparently healthy camels (97 and 50 from El-Bassatin and El-Warraq abattoirs respectively) for screening of Trypanosoma species infection. All samples were examined parasitologically by Giemsa stained blood smear and haematocrit centrifugation technique, serologically by card agglutination test (CATT) for detection of anti-trypanosomal antibodies and polymerase chain reaction (PCR) for DNA amplification with aspecific primers for detection of trypanozoan parasites. Out of 187 camels, 14 camels were positive by parasitological methods with a percentage of 7.49%, 21 positive samples were detected by anti- Trypanosoma antibodies using CATT with a percentage of 11.23%. Fourteen out of the positive blood samples by parasitological and serological techniques were used for PCR amplification. Thirteen out of 14 positive blood samples were PCR-positive and one was negative by using specific primers for T. evansi minicircle EVA1 and EVA2. The results of examined camels after using the polymerase chain reaction (PCR) for detection of DNA of trypanosomes in infected camels, showed 138 bp PCR product for the specific detection of Trypanosoma evansi. It was concluded that the use of PCR, beside parasitological and serological methods, is recommended for exact diagnosis in survey and control programmes ofTrypanosoma evansi.

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

The application of high resolution melting in the analysis of simple sequence repeat and single nucleotide polymorphism markers in a pea (Pisum sativum L.) population

The aim of this study was to verify the high resolution melting (HRM) method in the analysis of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in pea (Pisum sativum L.). A recombinant inbred line population, Carneval × MP1401, was tested for three SNP and 103 SSR markers. HRM analysis was conducted on a LightScanner 96 instrument with LC Green dye. The melting curve shape permitted two polymorphic genotypes to be distinguished. The results were confirmed by gel electrophoresis. Three SSR markers were sequenced and analysed by the melting prediction software. The results confirmed the presence of one polymerase chain reaction (PCR) product with two melting domains. Sequence tagged site (STS) markers produced specific products: Psat_EST_00189_01_1 (300 bp), Pis_GEN_18_2_1 (400 bp), Pis_GEN_7_1-2_1 (600 bp). Amplicons contained one, four and seven single nucleotide polymorphisms, respectively. Melting curve differences enabled the population genotyping except for Psat_EST_00189_01_1 where resolution was too low. Primers for Psat_EST_00189_01_1 were redesigned to obtain a shorter (100 bp) PCR product which increased the resolution. The number of SNPs and amplicon length are crucial for HRM resolution. The HRM method is fast and has a high throughput. The melting analysis of 96 samples takes less than 10 min. Agarose gel analysis confirmed the reliability of HRM, which eliminates laborious post-PCR analysis.

High Percentage of JAK2 exon 12 Mutation in Polycythemia Vera and Essential Thrombocythemia Among Populations of Qatar

AbstractAbstract 5064The chronic myeloproliferative Neoplasm (NPM) are clonal hematopoietic stem cell malignancies with 3 main subtypes: polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis. PV is characterized by increased RBC proliferation in the absence of erythropoietin and proliferation of myeloid lineages usually is noted, A gain-of-function mutation of Janus kinase 2 (JAK2) V617F, is identified in about 95% of patients with PV and about 50% of patients with essential thrombocytosis and idiopathic myelofibrosis.It has been shown that JAK2 exon 12 mutations can activate erythropoietin signaling pathways while these findings have been confirmed by many studies from Western countries, there are no reports from Asian countries in general and Arab countries in particular about the prevalence of the JAK2 exon 12 mutation in patients with PV and ET. In the present study, we determined the prevalence of JAK2V617F and JAK2 exon 12 mutations in patients with PV and ET in Qatar. Materials and MethodsWe enrolled patients with a diagnosis of PV and ET at National Centre for Cancer Care and Research in Qatar from January till June 2012. The diagnosis of PV and ET was established according to the 2008 World Health Organization criteria. The study included 82 patients. Clinical information and the CBC data at diagnosis were obtained from medical records. Pretreatment serum erythropoietin levels. Total DNA was isolated from buffy coat cells taken from peripheral blood using a kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Allele-specific polymerase chain reaction (PCR) was performed using 80 ng of genomic DNA as the template in a35-cycle PCR reaction at an annealing temperature of 58°C, as previously described. The mutant allele yields a 203-base-pair (bp) PCR product (sensitivity of mutant allele detection <1%).For exon 12 mutation screening, 80 ng of genomic DNA was amplified by specific primers designed to amplify a region of 453 bp containing the 128 bp of the exon 12 sequence of JAK2. PCR products were directly sequenced in both directions on an ABI 3730 DNA Analyzer using the BigDye Terminator Sequencing kit. ResultsWe examined the occurrence of JAK2V617F and JAK2 exon 12 mutations in a clinical cohort of 82 patients with polycythemia vera (PV) and Essential thrombocythemia (ET) Of which 42 patients had PV aged 25 to 53, 13 (31%) females and 29 (69 %) males and V617F mutation was detected in all of them exon 12 mutation was detected in 38 (90. 47%) patients. We found 2 different exon 12 mutations:3 N542-E543del, 1 F537-K539delinsL, and among 40 ET patients aged 25 to 59, 22 (55 %) males and 18 (45%) females, 35 patients (87. 5%) were JAK2 V617F and JAK 2 exon12 positive and 5 (12. 5%) were JAK2V617F as well as exon 12 negative patients with V617F and exon 12 mutations showed significantly higher WBC and platelet counts at diagnosis than patients with exon V617F mutation alone (P =. 021 and P =. 038, respectively). We report a surprisingly high incidence of exon 12 mutations in MPN patients with PVand ET in Qatar, a result quite different from reports in the Western literature (P =. 001). ConclusionOur data suggest that exon 12 mutation of JAK2 in patients with PV and ET may have an uneven geographic distribution. A clinical laboratory providing the V617F test alone may risk missing a substantial number of patients with PV in areas with a high incidence of exon 12 mutation. the importance of such associations may need further studies and evaluations. Disclosures:Yassin:Qatar National Research Fund: Patents & Royalties, Research Funding. Rafii El-Ayoubi:Qatar National Research Fund: Patents & Royalties, Research Funding. Al-Dewik:Qatar National Research Fund: Patents & Royalties, Research Funding.

Rapid identification of the medicinal plant Taraxacum formosanum and distinguishing of this plant from its adulterants by ribosomal DNA internal transcribed spacer (ITS) based DNA barcode

Original identification of medicinal plants is essential for quality control. In this study, the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA served as a DNA barcode and was amplified by allele-specific PCR. This approach was exploited to differentiate Taraxacum formosanum from five related adulterants. Using a set of designed PCR primers, a highly specific 223 bp PCR product of T. formosanum was successfully amplified by PCR. However, no similar DNA fragment was amplified from any of the other adulterants. This indicates that, our allele specific primers have high specificity and can accurately discriminate T. formosanum from its adulterant plants. Key words: Medicinal plant, polymerase chain reaction (PCR), authentication, Taraxacum formosanum, traditional Chinese medicinal, internal transcribed spacers 2 (ITS2).

A highly specific PCR assay for identification of goat (Capra hircus) meat

A highly specific single step polymerase chain reaction (PCR) is described for the detection of goat ( Capra hircus) meat. Goat-specific primers (DAF-01 and DGR-04) were designed against a conserved region of mitochondrial d-loop gene that yielded a 294 bp PCR product. Specificity of the primers was confirmed by testing them with DNA from other commonly used meat animal species, i.e., cattle, buffalo, sheep, swine and poultry. PCR amplification of target DNA with goat-specific primers was repeated 15 times, with consistent results observed. The specificity of goat-specific PCR provides a valuable tool for identification of goat meat and to avoid its fraudulent substitution and adulteration.

PCR-restriction endonuclease analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats, sheep, and cattle in Jordan

Paratuberculosis is an endemic disease and induces high economical losses in Jordan. There is no information available on genotypic variation of Mycobacterium avium subsp. paratuberculosis (MAP) isolated from animals in Jordan. In this study, we investigated 150 fecal samples from sheep, goats, and cattle for the presence of paratuberculosis using bacterial culture and polymerase chain reaction (PCR)-RFLP analysis of insertion sequence IS1311. Analysis of the results revealed that genotypic information from sheep, goat, and cattle could classify them into cattle or sheep strains. All culture isolates from cattle, 12.5% of the isolates from sheep, and 50% of the isolates from goats were cattle strain, while 87.5% of the isolates from sheep and 50% of the isolates from goats were sheep strain. Sequencing of the IS1311 268 bp PCR product from the three animal species confirmed the different MAP patterns.

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.

Characterization, detection and identification of transgenic chili pepper harboring coat protein gene that enhances resistance to cucumber mosaic virus

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

First report of a ‘Candidatus Phytoplasma asteris’‐related strain associated with a yellows disease of black pepper (Piper nigrum) in India

Black pepper ( Piper nigrum ) yellows is a newly recognized disease in Coorg (Kodagu) district of Karnataka State in southern India, where pepper is grown in 10 690 ha with a production of 2360 metric tonnes. Symptoms include yellowing and curling of the leaves. In the advanced stage, vines become yellow and slender with generalized decline and reduction in the yield noted. Based on observation of symptoms, an association of a phytoplasma with the disease was suspected. Leaf samples from vines with and without symptoms were collected from black pepper plants during December 2007 and January 2008. Total DNA was extracted as previously described (Adkar-Purushothama et al. , 2007), and assayed for phytoplasma infection through nested polymerase chain reaction (PCR) employing primer pairs P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (F2n/ R2) (Gundersen & Lee, 1996), for the amplification of phytoplasma 16S rDNA. DNAs extracted from ‘ Candidatus Phytoplasma asteris’-related strain AAY (group 16SrI) and ‘ Ca. Phytoplasma ulmi’-related strain EY1 (group 16SrV), maintained in periwinkle ( Catharanthus roseus ), were used as positive controls. Reaction mixtures containing DNA extracted from healthy periwinkle and reaction mixture without template DNA were used as negative controls. All samples from pepper plants with symptoms along with positive controls showed 1250 bp PCR products (F2n/R2 fragment), which were absent in symptomless samples. A representative phytoplasmal F2n/R2 fragment was cloned, sequenced and deposited in GenBank (FJ462798). The 16S rDNA nucleotide sequence of the phytoplasma identified in yellows-diseased black peppers [Coorg Black Pepper Yellows (CBPY) phytoplasma] shared > 99% sequence identity with that of the Sugarcane yellows phytoplasma type I (EU423900) from group 16SrI, and more than 98% with those of other related strains including AAY (X68373) and OY-M (AP006628). Moreover, CBPY phytoplasma showed 98% of identity with the BPP phytoplasma (AY823413), previously associated with phyllody of black pepper in India (Bhat et al ., 2006). This is the first molecular evidence for the association of a phytoplasma with a yellows disease of black pepper, and the identification of a ‘ Ca . Phytoplasma asteris’-related strain associated with such disease in India.

Fresh-frozen, optimal cutting temperature (OCT) compound-embedded bone marrow aspirates: a reliable resource for morphological, immunohistochemical and molecular examinations

The usefulness of fresh-frozen, optimal cutting temperature (OCT) compound-embedded (FFOE) bone marrow (BM) aspirates was evaluated as a reliable resource for morphological, immunohistochemical and molecular examinations. One hundred BM aspirates were collected in polypropylene tubes and immediately frozen for 2 h in a deep freezer. Frozen BM was transferred to a cryomold filled with OCT compound and the prepared samples were stored in a deep freezer. Histological examination and immunohistochemical staining, polymerase chain reaction (PCR), sequencing and reverse transcription (RT)-PCR were performed to evaluate the quality of the FFOE BM sections in 10% of randomly selected samples. FFOE BM sections revealed better morphologies than paraffin-embedded clot sections in haematoxylin and eosin staining because mature erythrocytes were removed during the staining process in frozen BM sections. Immunohistochemical staining for CD34 revealed excellent staining quality and oil red O staining showed that fat vacuoles in cells were well preserved. The quality of genomic DNA in FFOE BM sections was suitable for obtaining about 2000 bp PCR product for the human leucocyte antigen-A locus followed by direct sequencing of the sample, and the quality of total RNA was suitable for detection of BCR-ABL fusion transcript. FFOE BM aspirates are a reliable resource for various laboratory tests of diagnostic and research arenas.

Simple Multiplex PCR for Rapid Diagnosis of Sex of Ducks and Duck Embryos

Haunshi, S., Saxena, S.C., Pattanayak, A., Bandyopadhaya, S., Tyagi, A.K. and Bujarbaruah, K.M. 2008. Simple multiplex PCR for rapid diagnosis of sex of ducks and duck embryos. J. Appl. Anim. Res., 33: 117–120. A simple and rapid multiplex polymerase chain reaction (PCR) was developed for quick diagnosis of sex of duck and duck embryos. W chromosome specific DNA sequence was selected and primers were designed to amplify 335 bp fragment from female sex while 16S ribosomal sequence was selected to design primers to amplify 468 bp PCR products both in male and female sex as an internal control. Nucleotide sequences of W chromosome specific DNA fragments of Khaki Campbell and Indian Runner breeds of duck were found to be identical both in size and sequences. The study concluded that the protocol was successful in precisely identifying the gender of ducklings belonging to both Indian Runner and Khaki Campbell breeds of ducks and duck embryos.