Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis

2000 ◽  
Vol 76 (3) ◽  
pp. 221-228 ◽  
Author(s):  
H.S Nielsen ◽  
T Storgaard ◽  
M.B Oleksiewicz
2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.


2012 ◽  
Vol 47 (No. 10 - 11) ◽  
pp. 295-301 ◽  
Author(s):  
S. Indik ◽  
L. Valíček

Restriction fragment length polymorphism (RFLP) analysis of open reading frame 5 was developed for typing of Czech strains of porcine reproductive and respiratory syndrome virus (PRRSV). The set of restriction enzymes Acc I, Hae II and SnaB I allowed the differentiation of heterogeneous Czech strains of PRRSV clustered separately in the phylogenetic tree. The high-passage strain V-502 (164) was also differentiated from its parent strain V-502. The same restriction enzymes could distinguish the European-type vaccine strains Porcilis PRRS and Pyrsvac-183, registered inCzechRepublic, from the Czech field isolates. The published ORF5 nucleotide sequences allowed us to presume that it will also be possible to distinguish most of European field strains from vaccine strains. PCR-based RFLP analysis can become a valuable tool in epidemiological studies of PRRSV inEurope.


Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.


2003 ◽  
Vol 15 (4) ◽  
pp. 390-394 ◽  
Author(s):  
Dawn C. Hayes ◽  
Rebecca R. Anderson ◽  
Richard L. Walker

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.8S rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.8S rRNA gene and ITSR sequence results and PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.


2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.


1998 ◽  
Vol 10 (2) ◽  
pp. 140-144 ◽  
Author(s):  
Ronald D. Wesley ◽  
William L. Mengeling ◽  
Kelly M. Lager ◽  
Deborah F. Clouser ◽  
John G. Landgraf ◽  
...  

The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiating a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain from other North American field strains was investigated. Open reading frame 5 nucleotide sequence data of the vaccine virus, its parent strain VR-2332, and 22 other strains of PRRSV included in this study indicated that 3 restriction enzyme gel patterns characterize the vaccine virus and the parent strain genotype. The combined 3 RFLP patterns differentiate the vaccine and parent virus from other PRRSV strains. This test will be a valuable tool in epidemiologic studies and will be useful in identifying individual strains in cases of multistrain PRRSV infections.


2002 ◽  
Vol 14 (4) ◽  
pp. 343-347 ◽  
Author(s):  
Hugh Y. Cai ◽  
Hazel Alexander ◽  
Susy Carman ◽  
Dara Lloyd ◽  
Gaylan Josephson ◽  
...  

From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis. A total of 516 samples from 284 cases were RT-PCR positive for the PRRSV open reading frame (ORF) 7 sequence. The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 of PRRSV. Samples from 254 cases were typeable, yielding 34 different RFLP types. Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, 4 had a RFLP type shared by another commercial vaccine and a field strain. In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm. Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the specified time frame. In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged. There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV. There were 18 cases, consisting of 9 different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus.


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