The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.     

Molecular identification of hairtail species (Pisces: Trichiuridae) based on PCR-RFLP analysis of the mitochondrial 16S rRNA gene

A rapid PCR-RFLP analysis was designed to identify 3 closely related species of hairtails: Trichiurus lepturus, T. japonicus, and Trichiurus sp. 2, basing on partial sequence data (600 bp) of the mitochondrial DNA encoding the 16S ribosomal RNA (16S rRNA) gene. Restriction digestion analysis of the unpurified PCR products of these 3 species, using EcoRI and VspI endonucleases, generated reproducible species-specific restriction patterns showing 2 fragments (250 bp and 350 bp) for T. lepturus in EcoRI digestion and 2 fragments (196 bp and 404 bp) for T. japonicus in VspI digestion, whereas no cleavage was observed for Trichiurus sp. 2 in both EcoRI and VspI digestions. The PCR-RFLP technique developed in this study proved to be a rapid, reliable and simple method that enables easy and accurate identification of these 3 closely related species of the genus Trichiurus.     

Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene

This study amplified the mitochondrial 16S rRNA gene using polymerase chain reaction (PCR) with a template of total DNA from muscle tissues of nine pufferfish species collected from the coastal area of Okinawa Islands in Japan: Pleuranacanthus sceleratus, Triodon macropterus, Chelonodon patoca, Sphoeroides pachygaster, Arothron hispidus, A. stellatus, A. manilensis, A. mappa, and A. nigropunctatus. Then nucleotide sequence encoding a partial region of the 16S rRNA gene was compared among species. The sequenced fragment was also used to select restriction enzymes, yielding species-specific restriction fragment length polymorphisms (RFLP). The sequence of the segment of the 16S rRNA gene consisted of about 615 nucleotides and showed interspecies variations in the targeted region. After calculation of corresponding RFLP-patterns of nine species investigated with suitable restriction enzymes, three restriction enzymes – BanII, DdeI, and NlaIII – were found to be sufficient for identification of all nine species. Successful testing of this methodology in frozen and heated food samples suggests its utility for pufferfish species authentication in food products.     

双壳纲贝类18S rRNA基因序列变异及系统发生

双壳纲贝类栖息于环境多变的海域,是一个形态学和生态学都具有多样性的类群,清晰而可靠的进化关系对于养殖与相关种类的管理具重要意义。然而,目前对双壳类宏观分子系统学研究的报道较少。研究用18S rRNA基因(18S)分析了双壳类3个亚纲贝类的系统发育关系。从GenBank下载帘蛤目、海螂目、贻贝目、胡桃蛤目、蚶目、珍珠贝目6个目94个种类的18S全/部分序列107个,通过ClustalX软件进行序列比对, 用MEGA4.1软件和PHyML软件计算遗传距离, 构建系统发育树, 研究了双壳类18S变异规律及其在系统发生研究中的应用。结果显示18S有插入/缺失序列, 存在长度多态性。序列比对显示有5段约30 70bp的保守区, 4段约130 550bp的高变区。碱基组成平均为T:24.4%, C:23.6%, A:24.5%, G:27.5%。G+C含量为51.1%。在1796个比对位点中, 变异位点占31.7%, 简约信息位点占24.0%。目内科间遗传距离为0.003 0.043, 目间遗传距离为0.026 0.093。NJ树和ML树显示贻贝目、珍珠贝目、胡桃蛤目、蚶目和海螂目的缝栖蛤科先分别聚为支持率很高(BPN=94 100)的单系支, 后聚为一大支(BPN=100)。蛤蜊科与帘蛤目的其他科分离形成一置信度很高的单系支(BPN=93)。帘蛤科种类聚为置信度较低(BPN=60)的一支。海螂目、帘蛤目的种类没能完全聚到所属支系, 彼此嵌套,缝栖蛤科的种类从海螂目中分离出来。18S资料揭示帘蛤目的蛤蜊科、海螂目的缝栖蛤科已经进化为独立的支系。     

The identification of <Emphasis Type="Italic">Cryptosporidium</Emphasis> species in Isfahan,Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that causes diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, imunocompromised patients, and high-risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of the 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession no. DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession no. DQ520952). We also found a new genotype infecting both human and cattle samples (GenBank accession no. DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal and human cycle. Published in Russian in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 5, pp. 934–939. The article was translated by the authors.     

Molecular phylogeny of Sarcocystis fayeri (Apicomplexa: Sarcocystidae) from the domestic horse Equus caballus based on 18S rRNA gene sequences and its prevalence

Sarcocystosis is a parasitic disease caused by an intracellular protozoan parasite Sarcocystis belonging to the phylum Apicomplexa. These parasites have a requisite two-host life cycle. Recently, there are many Sarcocystis species that identified morphologically. In the present study, diaphragmatic muscle samples from the domestic horse (Equus caballus) were examined for Sarcocystis infection. The natural infection with sarcocysts was recorded to be 62·5% for only microcysts in the infected muscles. Molecular analysis using the 18S rRNA gene was conducted to swiftly and accurately identify the recovered species. Studies on the expression of the 18S rRNA gene have confirmed that the present parasite isolates belong to the Sarcocystis genus. The sequence data showed significant identities (>80%) with archived gene sequences from species within the Sarcocystidae family, and a dendrogram showing the phylogenetic relationship was constructed. The most closely related species were the previously described Sarcocystis fayeri and Sarcocystis bertrami. The current data showed that the present species was identified as S. fayeri and deposited in GenBank (accession number MF614956.1). This study highlights the importance of the genetic data in the exact taxonomy within sarcocystid species.     

16S rRNA sequencing in routine bacterial identification: a 30-month experiment

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness of this method for identifying a range of bacteria in a clinical laboratory under routine conditions. Over a period of 30 months, 683 isolates were obtained from clinical specimens, sequenced and analysed. For 568 of these isolates (83.1%), the sequence provided species level identification. For 108 isolates (15.8%), the identification was limited to the genus level, and for 7 isolates (1%), the sequence remained unidentifiable by 16S rDNA sequence analysis. For the isolates identified only to the genus level, the 16S rDNA approach failed to identify bacteria to the taxonomic level for 3 reasons: failure to differentiate between species in 72 isolates (66%), the lack of any closely related sequence in the database for 15 isolates (13.8%) and the presence of more than 1% of undetermined position in the sequence for 13 isolates (12%).     

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing.     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Unusual intraindividual variation of the nuclear 18S rRNA gene is widespread within the Acipenseridae

Significant intraindividual variation in the sequence of the 18S rRNA gene is unusual in animal genomes. In a previous study, multiple 18S rRNA gene sequences were observed within individuals of eight species of sturgeon from North America but not in the North American paddlefish, Polyodon spathula, in two species of Polypterus (Polypterus delhezi and Polypterus senegalus), in other primitive fishes (Erpetoichthys calabaricus, Lepisosteus osseus, Amia calva) or in a lungfish (Protopterus sp.). These observations led to the hypothesis that this unusual genetic characteristic arose within the Acipenseriformes after the presumed divergence of the sturgeon and paddlefish families. In the present study, a survey of nearly all Eurasian acipenseriform species was conducted to examine 18S rDNA variation. Intraindividual variation was not found in the polyodontid species, the Chinese paddlefish, Psephurus gladius, but variation was detected in all Eurasian acipenserid species. The comparison of sequences from two major segments of the 18S rRNA gene and identification of sites where insertion/deletion events have occurred are placed in the context of evolutionary relationships within the Acipenseriformes and the evolution of rDNA variation in this group.     

Characterization and identification of compost bacteria based on 16S rRNA gene sequencing

The aim of this study was to isolate and characterize bacteria from the compost of fruit and vegetable waste (FVW) for plant growth-promoting (PGP) activities and investigate the pro-active influence of bacterial isolates on wheat growth. Fourteen bacterial strains (RHC-1 to RHC-14) were isolated and purified in tryptic soya agar (TSA). In addition to being biochemically characterized, these bacterial strains were also tested for their PGP traits, such as phosphate (P)-solubilization, nifH gene amplification, indole-3-acetic acid (IAA) quantification and the production of ammonia, oxidase and catalase. Based on 16S rRNA gene sequencing, these bacterial strains were identified as belonging to species of Bacillus, Lysinibacillus, Lysobacter, Staphylococcus, Enterobacter, Pseudomonas and Serratia. All bacterial strains solubilized tri-calcium phosphate and produced IAA. Two bacterial strains RHC-8 (Enterobacter sp.) and RHC-13 (Pseudomonas sp.) solubilized the maximum amount of tri-calcium phosphate, i.e. 486 and 464 μg/ml, respectively. P-solubilization was associated with a significant drop in the pH of the broth culture from an initial pH of 7 to pH 4.43. In addition to P-solubilization and IAA production, six bacterial strains also carried the nifH gene and were further evaluated for their effect on wheat (Triticum aestivum) growth under controlled conditions. All six bacterial strains enhanced wheat growth as compared to uninoculated control plants. Two of the bacterial strains, RHC-8 and RHC-13, identified as Enterobacter aerogenes and Pseudomonas brenneri, respectively, were assessed as potential PGP rhizobacteria due to exhibiting characteristics of four or more PGP traits and enhancing wheat growth though their specific mechanism of action.     

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.      

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.     

Cloning and sequence analysis of the 18 S ribosomal RNA gene of tomato and a secondary structure model for the 18 S rRNA of angiosperms

Summary The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (ycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 by long with a G+C content of 49.6%. Its sequence exhibits 94%–96% positional identity when it is colinearly aligned with the previously reported sequences of the 17–18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.     

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

九种绢丝昆虫线粒体12S rRNA基因的序列特征和系统发育分析

为探讨鳞翅目中绢丝昆虫之间的系统发育关系和分子进化特征,本研究测定了中国柞蚕Antheraea pernyi野生型和放养型的线粒体12S rRNA基因的部分序列,结合来自GenBank数据库的17条序列,对总共9种绢丝昆虫（2科3属）的12S rRNA基因序列进行了分析。利用软件MEGA 3.1进行碱基组成、变异位点的统计和分子进化分析,分别用类平均聚类法（UPGMA）、邻接法（NJ）、最小进化法（ME）、最大简约法（MP）重建系统发生树。测定的中国柞蚕野生型的12S rRNA基因序列（427 bp）与放养型“豫早1号”的序列完全一致。序列对齐后共鉴定80个变异位点,50个简约信息位点。碱基组成分析显示在科属间具有明显差异,AT含量蚕蛾科高于大蚕蛾科;在A和T碱基的使用上,大蚕蛾科偏好使用T,而蚕蛾科则偏好使用A。与动物中常见的以转换为主的碱基替换模式不同,所分析的9种昆虫中除桑蚕属内部为转换与颠换基本一致外,其余物种间均是颠换多于转换。进化分析支持柞蚕属、樗蚕属和桑蚕属的单系。基于UPGMA法的进化树支持琥珀蚕是柞蚕属的较原始类型,而NJ、ME和MP法则支持印度柞蚕是较原始的类型,因此,柞蚕属种间的进化关系尚需进一步研究。     

Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study,we determined species-specific variations by analyzing the mitochondrial 12S rRNA gene sequence variation(～440 bp) in 17 newly obtained sequences and 90 published cattle,yak,buffalo,goat,and pig sequences,which represent 62 breeds and 17 geographic regions.Based on the defined species-specific variations,two endonucleases,Alu I and Bfa I,were selected for species authentication using raw meat/tissue samples and the PCR-RFLP method.Goat and pig were identified using the Alu I enzyme,while cattle,yak,and buffalo were identified by digestion with Bfa I.Our approach had relatively high detection sensitivity of cattle DNA in mixed cattle and yak products,with the lowest detectable threshold equaling 20% of cattle DNA in a mixed cattle/yak sample.This method was successfully used to type commercial beef jerky products,which were produced by different companies utilizing various processing technologies.Our results show that several yak jerky products might be implicated in commercial fraud by using cattle meat instead of yak meat.