Species-specific Polymerase Chain Reaction Method Research Articles

Detection of Tirmania pinoyi in Roots of Inoculated Cistaceae Plant Species by Nested Polymerase Chain Reaction

Desert truffles are edible hypogeous fungi, which grow in arid and semiarid areas of the Mediterranean region. They play an important role in the maintenance of rangelands by preventing erosion and desertification. In recent years, rangelands have experienced serious crises due to global warming and groundwater scarcity. One of the ways to improve rangelands is to use desert truffles as part of conservation or restoration efforts. For these reasons it is important to study the mycorrhizal association of these fungi with rangeland plants. For the first time, we investigated the mycorrhizal association of the desert truffle (Tirmania pinoyi) with the perennial plant species Helianthemum lippii, Helianthemum almeriense, Cistus laurifolius, and Cistus ladanifer. We inoculated all four plant species with T. pinoyi and found the presence of an ectendomycorrhizal association with varying degrees of sheath development. Our results shows that H. lippii is a suitable plant host for T. pinoyi, and the mycorrhization rate and relative mycorrhizal dependency in this native plant were about 90% and 57.85%, respectively, and higher than the other three plant species. The effect of the mycorrhizal fungus on host growth (root weight, root height, shoot weight, shoot height, plant height, and plant weight) was statistically significant compared with noninoculated plants. To detect the roots colonized by T. pinoyi, a polymerase chain reaction (PCR)-based diagnostic method was developed with the species-specific primer pairs FTiPi and RTiPi designed from the sequence of the internal transcribed space regions (ITS1 and ITS2). The specificity of the primers was verified by PCR analysis of DNA from T. pinoyi specimens and other desert truffles. The method detected T. pinoyi in inoculated root plants, and no cross-reactions were observed with any other tested desert truffles. This species-specific PCR method is suitable for quick, simple, and reliable detection of T. pinoyi mycorrhizas.

PCR analysis for meat products authenticity - detection of horse meat

Food adulteration regarding species origin of meats is a common problem in the meat products sector. With regard to horse meat, its undeclared use in food products is not only a fraud, but could present a health risk since is often associated with the presence of the veterinary drug phenylbutazone in meat products. Therefore, it is important to use reliable methods for authentication of meat products regarding their species composition, which are applicable to complex food matrices. Polymerase chain reaction (PCR) with species-specific primers remains the most widely used analytical approach to detect species-related food adulteration due to its high sensitivity and specificity. The aim of the present study was to establish the authenticity of 20 different meat products on the Bulgarian market without declared horse meat content by using a species-specific PCR method. The specificity test of the PCR method used showed no amplification of DNA from beef and pork. A detection limit of 0.01% horse DNA in three-component meat mixtures was established for the PCR method. The PCR method enabled detection of undeclared presence of horse meat in 25% of the analyzed meat products, which demonstrates the need for strict control regarding authenticity in the meat food chain.

새우에서 분리된 Vibrio species 동정을 위한 VITEK 2 system방법과 species-specific PCR방법 비교 평가

Vibrio is a genus of Gram-negative, curved, halophilic, and non-spore-forming bacteria. Some of the Vibrio species, such as V. cholerae and V. parahaemolyticus, often contaminate seafood products and occasionally cause human diseases when the seafood products are ingested. A total of 24 Vibrio strains were isolated from shrimp samples on Thiosulphate citrate bile salt sucrose (TCBS) media in this study. All of the 24 isolates were confirmed to belong to the genus Vibrio by using 16S rRNA gene sequence analyses. Vitek 2 system and species-specific poly- merase chain reaction (PCR) methods were used to further identify a total of 29 Vibrio strains at the species level, including the 24 shrimp Vibrio isolates and five Vibrio reference strains. The specificities of the two methods to iden- tify Vibrio strains at the species level were compared in this study. The species-specific PCR method was designed to detect five different Vibrio species, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio algi- nolyticus, and Vibrio mimicus. From the 24 Vibrio shrimp isolates, the Vitek 2 system method could identify 15 (62.5%) strains as Vibrio species and 7 (29.2%) strains as non-Vibrio species, but could not identify the rest 2 (8.3%) strains. But species-specific PCR method could identify 16 (66.7%) strains as Vibrio species and could not identify the rest 8 (33.3%) strains. Among the 24 Vibrio shrimp strains, these two methods could unanimously identify 7 (7/24, 29.2%) strains (2 V. parahaemolyticus, 4 V. alginolyticus, and 1 V. mimicus). Considering that such different identifi- cation results were obtained using the two different methods in this study, identification method for Vibrio species must be carefully chosen.

Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilis by PCR.

A polymerase chain reaction (PCR) assay was developed for discrimination of Bacillus subtilis from other members of B. subtilis group as well as rapid identification from environmental samples. Primers ENIF and EN1R from endoglucanase gene were used to amplify a1311 bp DNA fragment. The specificity of the primers was tested with seven reference strains and 28 locally isolated strains of endoglucanase positive Bacillus species. The PCR product was only produced from B. subtilis. The results demonstrated high specificity of two oligonucleotides for B. subtilis. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of B. subtilis. To our knowledge this is the first report of a B. subtilis specific primer set.

Identification of 11 sea cucumber species by species-specific PCR method

A species-specific PCR (polymerase chain reaction) method was developed to identify the 11 sea cucumber species, even in highly processed food samples such as frozen or dried sea cucumbers (beche-de-mer). Universal primers were used for the amplification of 16S rRNA gene in each species. The alignment of the obtained sequences was the basis for the design of 11 species-specific reverse primers located in the 16S rRNA gene region. The developed species-specific PCR method was successfully applied to authenticate species of commercial sea cucumber products. Therefore, this method really provides a simple, fast and reliable protocol for the accurate identification of sea cucumber species, and also can be very useful for correct labeling of commercial products, traceability, and protection of the consumer's rights.

Application of species-specific PCR for the identification of dried bonito product (Katsuobushi)

The species-specific PCR (polymerase chain reaction) method was developed to identify the species of dried bonito product (Katsuobushi) produced from Euthynnus pelamis, E. affinis, Auxis rochei, A. thazard, and Sarda orientalis. The 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five related Scombridae species were established, and then five pairs of species-specific primer were designed to amplify short length fragments among bonito species. The developed species-specific PCR method was successfully applied to authenticate species of commercial dried bonito products. Hence, this method really provided a useful and academic technique to identify the sources of bonito product.

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque.

Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.