Improving PCR detection of prey in molecular diet studies: importance of group‐specific primer set selection and extraction protocol performances

2012 ◽  
Vol 13 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Diane Zarzoso‐Lacoste ◽  
Emmanuel Corse ◽  
Eric Vidal
Keyword(s):  
Pcr Detection ◽  
Specific Primer ◽  
Extraction Protocol ◽  
Group Specific Primer

scholarly journals PCR-Based Identification of Pandemic GroupVibrio parahaemolyticuswith a Novel Group-Specific Primer Pair

2004 ◽  
Vol 48 (10) ◽  
pp. 787-790 ◽  
Author(s):  
Masatoshi Okura ◽  
Ro Osawa ◽  
Atsushi Iguchi ◽  
Michihiro Takagi ◽  
Eiji Arakawa ◽  
...  
Keyword(s):  
Specific Primer ◽  
Group Specific Primer

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method

2005 ◽  
Vol 52 (1-2) ◽  
pp. 85-91 ◽  
Author(s):  
Y. Yu ◽  
C. Lee ◽  
S. Hwang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
Quantitative Real Time Pcr ◽  
Community Structures ◽  
Specific Primer ◽  
Anaerobic Processes ◽  
Promising Tool ◽  
Pcr Method ◽  
Group Specific Primer

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

scholarly journals PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

2001 ◽  
Vol 8 (3) ◽  
pp. 499-502 ◽  
Author(s):  
Hideaki Ikejima ◽  
Shusaku Haranaga ◽  
Hiromu Takemura ◽  
Tsutomu Kamo ◽  
Youichi Takahashi ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Chlamydia Pneumoniae ◽  
Detection Method ◽  
Pcr Detection ◽  
Standard Protocol ◽  
Extraction Protocol ◽  
Highly Effective ◽  
Commercial Kit

ABSTRACT Since current studies indicate the possible involvement ofChlamydia pneumoniae in the pathogenesis of multiple sclerosis (MS), demonstration of C. pneumoniae in the cerebrospinal fluid (CSF) of patients with MS is highly desirable. However, there is controversy concerning the detection of C. pneumoniae in CSFs from MS patients due to the lack of a standard protocol for extraction and detection of C. pneumoniae DNA. In this regard, we attempted to establish a highly effective extraction protocol for C. pneumoniae DNA from CSFs utilizing a commercial kit and a PCR detection method. The extraction and PCR detection protocol established in this study succeeded in detecting as few as 20 C. pneumoniae organisms in 200 μl of mock CSF. The use of this protocol to detect C. pneumoniae DNA in CSFs revealed that 68% of CSF samples obtained from patients with MS were positive (11 out of 16 samples) for chlamydia DNA. Thus, the protocol established here is sensitive enough to detect chlamydia DNA from CSFs and can be used by other laboratories for evaluation of the presence of chlamydiae in CSFs because the protocol is based on the use of a commercial kit.

scholarly journals Rapid PCR-based Detection of Phytoplasmas from Infected Plants

HortScience ◽  
2003 ◽  
Vol 38 (6) ◽  
pp. 1134-1136 ◽  
Author(s):  
Yonghong Guo ◽  
Zong-Ming Cheng ◽  
James A. Walla
Keyword(s):  
Sodium Hydroxide ◽  
Cross Sections ◽  
Infected Plant ◽  
Pcr Amplification ◽  
Green Ash ◽  
Specific Primer ◽  
Plant Materials ◽  
Dna Preparation ◽  
Group Specific Primer ◽  
Triton X

Five simplified DNA preparation procedures for polymerase chain reaction (PCR) amplification were tested for detection of phytoplasmas from infected herbaceous and woody plants. Thin freehand cross-sections made from infected plant tissues and stored in acetone were used as sources for DNA preparation. The tissue sections were treated by: 1) grinding in sodium hydroxide; 2) sonicating in water; 3) microwaving in water; 4) boiling in sodium hydroxide; or 5) placing directly in PCR tube. PCR amplification was performed with a universal phytoplasma-specific primer pair in a reaction buffer containing 0.5% (v/v) Triton X-100, 1.5 mm magnesium chloride, and 10 mm Tris-HCl. All five procedures provided phytoplasmal template DNA for successful PCR amplification from infected herbaceous plants {periwinkle [Catharanthus roseus (L.) G. Don (periwinkle)], carrot (Daucus carota L.), maize (Zea mays L.)}, while the grinding, microwaving, and boiling procedures also allowed positive amplification from a woody plant [green ash (Fraxinus pennsylvanica Marsh.)]. The quality of the resulting DNA was adequate for subsequent identification of the aster yellows and ash yellows phytoplasmas through nested-PCR using phytoplasma group-specific primer pairs. These methods provide remarkable savings in labor and materials, making disease testing and indexing of plant materials much more attractive.

scholarly journals First Report of Streptocarpus flower break virus in Streptocarpus in the United States

Plant Disease ◽  
2007 ◽  
Vol 91 (10) ◽  
pp. 1361-1361 ◽  
Author(s):  
H. R. Pappu ◽  
K. B. Druffel
Keyword(s):  
United States ◽  
Genomic Sequence ◽  
The United States ◽  
Genomic Region ◽  
Rt Pcr ◽  
Specific Primer ◽  
Sequence Comparisons ◽  
First Report ◽  
Group Specific Primer ◽  
Free Material

Streptocarpus flower break virus (SFBV) belongs to the genus Tobamovirus and was described from naturally infected streptocarpus plants in 1995 (2). The complete genomic sequence was recently reported (1). Prominent symptoms include flower breaking while foliar symptoms are often lacking. In March 2007, four streptocarpus plants (cv. Indigo Dream) from San Diego County, CA were tested for the presence of SFBV by ELISA and reverse transcription (RT)-PCR. Symptoms suggestive of a virus infection were not present on these mother plants at the time of sampling. ELISA with SFBV-specific antiserum showed that all samples were infected with SFBV. The ELISA results were verified by RT-PCR followed by cloning and sequencing. Two sets of primer pairs were used in separate RT-PCR tests. One was a degenerate tobamovirus group-specific primer pair and the second primer pair was specific to SFBV (1). The tobamovirus group-specific primer pair consisted of Tob Uni1, 5′-ATT TAA GTG GAG GGA AAA CCA CT-3′ and Tob Uni2, 5′-GTY GTT GAT GAG TTC GTG GA-3′. The SFBV-specific primers were SFBVcpF: 5′-AAA ATG TCG TAC GTG GTG GT and SFBVcpR: 5′-ACC CAC AGA ACT TCC TTC AA-3′ (1). PCR amplicons of the expected size (686 bp for the tobamovirus group-specific primer pair and 562 bp for the SFBV-specific primer pair) were obtained for each primer pair. The positive PCR test using the SFBV-specific primer pair confirmed the presence of SFBV. To further verify the identity of the virus, the amplicons obtained with each primer pair were separately cloned and sequenced. At least two clones for each amplicon were sequenced in both directions. Sequence comparisons with those available in GenBank showed 98% sequence identity with the corresponding genomic region (GenBank Accession No. NC_008365) of SFBV (1). To our knowledge, this is the first report of SFBV in the United States and it highlights the need for testing for this virus to ensure propagation and distribution of virus-free material. References: (1) C. Heinze et al. Arch. Virol. 151:763, 2006. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 101:311, 1995.

scholarly journals Specific Primer Sets for RT-PCR Detection of Major RNA Viruses of Tomato Plants in Korea

2017 ◽  
Vol 23 (2) ◽  
pp. 193-201
Author(s):  
Jun-Sung Shin ◽  
Jung-Heon Han ◽  
Yu-Ju Shin ◽  
Hae-Ryun Kwak ◽  
Hong-Soo Choi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Specific Primer ◽  
Tomato Plants ◽  
Primer Sets
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