scholarly journals A Multiplex PCR for the Detection of Phytophthora nicotianae and P. cactorum, and a Survey of Their Occurrence in Strawberry Production Areas of Japan

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1270-1278 ◽  
Author(s):  
Mingzhu Li ◽  
Takahiro Asano ◽  
Haruhisa Suga ◽  
Koji Kageyama
Keyword(s):  
Multiplex Pcr ◽  
Protein Gene ◽  
Phytophthora Nicotianae ◽  
Soilborne Pathogens ◽  
Chain Reaction ◽  
Specific Primers ◽  
Phytophthora Spp ◽  
Polymerase Chain ◽  
Species Specific ◽  
Production Areas

We aimed to simultaneously detect two pathogens causing strawberry diseases, Phytophthora nicotianae and P. cactorum, by multiplex polymerase chain reaction (PCR), and to survey their occurrence in the main strawberry production areas of Japan. Due to the need to combine different primer pairs for multiplex PCR and the low specificity of published specific primers for P. nicotianae and P. cactorum, new species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions of ribosomal DNA and the ras-related protein gene Ypt1, respectively. Specificity of the designed primers was demonstrated using 68 isolates, including Phytophthora spp., Pythium spp., and other soilborne pathogens. Multiplex PCR discriminated between P. nicotianae and P. cactorum in DNA mixtures of mycelia of the two species. Moreover, both species were detected in artificially and naturally infested soils, indicating that these markers can be used in diagnosis of strawberry diseases. For investigation of the geographic distribution of the two pathogens in Japan, soil samples were collected in 89 strawberry fields from eight prefectures (Gifu, Saga, Nara, Tochigi, Chiba, Shizuoka, Yamanashi, and Hokkaido) of Japan. The method that was developed was successfully applied to survey P. nicotianae and P. cactorum, and distribution of the two pathogens in strawberry plantings in Japan was determined.

Microsporidial infections due toEncephalitozoon intestinalisin non-HIV-infected patients with chronic diarrhoea

2011 ◽  
Vol 140 (10) ◽  
pp. 1773-1779 ◽  
Author(s):  
J. YAKOOB ◽  
Z. ABBAS ◽  
M. ASIM BEG ◽  
W. JAFRI ◽  
S. NAZ ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Stool Examination ◽  
Chronic Diarrhoea ◽  
Healthy Controls ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Species Specific

SUMMARYWe determined the prevalence of microsporidiaEnterocytozoon(Ent.)bieneusiandEncephalitozoon(E.)intestinalisinfection in patients with chronic diarrhoea and hepatocellular carcinoma (HCC). A total of 330 stool samples were examined from 171 (52%) patients with chronic diarrhoea, 18 (5%) with HCC while 141 (43%) were controls. Stool microscopy, polymerase chain reaction (PCR) with species-specific primers forEnt. bieneusiandE. intestinalisand sequencing were carried out. Microsporidia were found by trichrome staining in 11/330 (3%) andE. intestinalisby PCR in 13/330 (4%) whileEnt. bieneusiwas not detected. PCR forE. intestinaliswas positive in 8/171 (5%) stool samples from patients with chronic diarrhoea, 2/141 (1·4%) samples from healthy controls and in 3/18 (17%) samples from patients with HCC. In the chronic diarrhoea group,E. intestinaliswas positive in 4/171 (2·3%) (P=0·69) stool samples compared to 2/18 (11%) (P=0·06) in the HCC group and 2/141 (1·4%) from healthy controls.E. intestinalisinfection was significantly associated with chronic diarrhoea and HCC in these patients who were negative for HIV. Stool examination with trichrome or species-specific PCR for microsporidia may help establish the cause of chronic diarrhoea.

a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2983-2983
Author(s):  
Silvia Gimondi ◽  
Alessandra Cavanè ◽  
Antonio Vendramin ◽  
Giulia Biancon ◽  
Paolo Corradini ◽  
...  
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Residual Disease ◽  
Chain Reaction ◽  
B Cell Malignancies ◽  
Specific Primers ◽  
Namalwa Cell ◽  
Polymerase Chain ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

Detection of cow milk paneer in mixed/buffalo milk paneer through conventional species specific Polymerase Chain Reaction

2016 ◽  
Author(s):  
Tanmay Hazra ◽  
Vivek Sharma ◽  
Rekha Sharma ◽  
S. De ◽  
Sumit Arora ◽  
...  
Keyword(s):  
Buffalo Milk ◽  
Cow Milk ◽  
Market Demand ◽  
Chain Reaction ◽  
Specific Primers ◽  
Mt Dna ◽  
Milk Adulteration ◽  
D Loop ◽  
Polymerase Chain ◽  
Species Specific

Due to higher market demand of buffalo milk paneer, lower price cow milk is often adulterated with higher cost buffalo milk for preparation of paneer. Till date no rapid technique is available in market to ensure that paneer is made from buffalo milk. Currently a PCR based method has been developed to authenticate the buffalo milk paneer. DNA was isolated from paneer by DNeasy Mericon food kit. A set of bovine specific primers (P1) targeting D-loop (displacement loop) of mt- DNA was selected and standardized to amplify cow DNA resulted 126bp amplicon. Using this PCR based approach even upto 1% level of cow milk adulteration in buffalo milk paneer could be detected.

scholarly journals A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

2003 ◽  
Vol 93 (7) ◽  
pp. 822-831 ◽  
Author(s):  
Ping Kong ◽  
Chuanxue Hong ◽  
Steven N. Jeffers ◽  
Patricia A. Richardson
Keyword(s):  
Polymerase Chain Reaction ◽  
Irrigation Water ◽  
Phytophthora Nicotianae ◽  
Detection Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Polymerase Chain Reaction ◽  
Wide Range ◽  
Polymerase Chain ◽  
Species Specific

Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/μl. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

Persistence of human mitochondrial DNA throughout the development to the blastocyst of mouse zygotes microinjected with human mitochondria

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 279-283 ◽  
Author(s):  
V.B. Vasilyev ◽  
V.A. Sokolova ◽  
A.V. Sorokin ◽  
M.G. Bass ◽  
N.I. Arbuzova ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Hepg2 Cell Line ◽  
Chain Reaction ◽  
Specific Primers ◽  
Human Mitochondrial Dna ◽  
Mouse Zygotes ◽  
Polymerase Chain ◽  
Species Specific

The conditions for transfer of human mitochondria into fertilised mouse ova were elaborated. Species-specific primers were designed to discriminate human mitochondrial DNA (mtDNA) and the endogenous mtDNA in the preimplantation embryos. Human mitochondria isolated from the HepG2 cell line were microinjected into murine zygotes, and the latter cultured for 96 h to the blastocyst stage. The polymerase chain reaction allowed the detection of human mtDNA at every stage of embryo cleavage. In some cases a clear disparity in distribution of human mtDNA among blastomeres was evident.

scholarly journals Morphological and Molecular Identification of Fusarium oxysporum Sch. Isolated From Guava Wilt in Bangladesh

2012 ◽  
Vol 41 (1) ◽  
pp. 49-54 ◽  
Author(s):  
M Zakir Hussain ◽  
MA Rahman ◽  
Mohammad Nurul Islam ◽  
MA Latif ◽  
MA Bashar
Keyword(s):  
Fusarium Oxysporum ◽  
Psidium Guajava ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Species Identity ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Serious Disease ◽  
Species Specific

Wilt of guava plants (Psidium guajava L.) is a serious disease in Bangladesh. Sixteen isolates of Fusarium oxysporum Sch. were collected from the root and stem fragments of guava plants growing in six districts of Bangladesh. Species identity was based on the colony character, nature of conidiogenous cell, morphology of microconidia, macroconidia and chlamydospores. Eleven isolates were confirmed as F. oxysporum through polymerase chain reaction (PCR) using species specific primers designed from the conserved regions of 18S rRNA gene. DOI: http://dx.doi.org/10.3329/bjb.v41i1.11082 Bangladesh J. Bot. 41(1): 49-54, 2012 (June)

scholarly journals Molecular identification of Ditylenchus destructor nematode with PCR Species-Specific primers in the Moscow region

2019 ◽  
Vol 14 (4) ◽  
pp. 430-436 ◽  
Author(s):  
Niloufar Mahmoudi ◽  
Yousef Naserzadeh ◽  
Elena N Pakina ◽  
Liudmila A Limantceva ◽  
Davoud Kartuli Nejad
Keyword(s):  
Solanum Tuberosum L ◽  
Nematode Species ◽  
Moscow Region ◽  
Molecular Technique ◽  
Chain Reaction ◽  
Specific Primers ◽  
The Russian Federation ◽  
Polymerase Chain ◽  
Species Specific ◽  
Ditylenchus Destructor

Potato ( Solanum tuberosum L.) is one of the most vital food and industrial crop and Ditylenchus destructor is an influential pathogenic potato nematode and is quarantine pest in many states and territories. As a result, the polymerase chain reaction (PCR) protocol was optimized to identify Ditylenchus destructor reliably and rapidly. The species-specific internal transcribed spacer (ITS) was used as the primer of the D. destructor ribosomal DNA gene. Some populations of this species from the Moscow region in the Russian Federation were investigated through species-specific primer PCR. New sequence from ITS-rRNA was deposited in the GenBank under accession number MN122076. The current molecular technique is more appropriate to distinguishing of nematode species, since it is practical, fast and precise.

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