Direct sequencing of the PCR amplified SSU rRNA gene ofEntamoeba disparand the design of primers for rapid differentiation fromEntamoeba histolytica

Parasitology ◽  
1996 ◽  
Vol 112 (4) ◽  
pp. 363-369 ◽  
Author(s):  
S. Novati ◽  
M. Sironi ◽  
S. Granata ◽  
A. Bruno ◽  
S. Gatti ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Sensu Stricto ◽  
Ssu Rdna ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
Data Bases ◽  
Entamoeba Dispar ◽  
Isoenzyme Analysis

SUMMARYSince 1993, strains ofEntamoeba histolytica sensn latohave been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains toE. histolytica sensu stricto, the non-pathogenic strains toEntamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases forE. histolytica, only a partial sequence has been published forE. dispar. Here we report a SSU rDNA sequence forE. dispar. Compared to those ofE. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from bothE. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphicDdeI restriction site which allows, after cleavage of the fragment,E. histolyticaandE. disparto be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

scholarly journals Quantitative Measure of Small-Subunit rRNA Gene Sequences of the Kingdom Korarchaeota

1998 ◽  
Vol 64 (12) ◽  
pp. 5064-5066 ◽  
Author(s):  
Clifford F. Brunk ◽  
Nicole Eis
Keyword(s):  
Internal Standard ◽  
Pcr Amplification ◽  
Quantitative Measure ◽  
Pcr Primers ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequence ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Rdna Sequences

ABSTRACT Comparative PCR amplification of small-subunit (SSU) rRNA gene (rDNA) sequences indicates substantial preferential PCR amplification of pJP27 sequences with korarchaeote-specific PCR primers. The coamplification of a modified SSU rDNA sequence can be used as an internal standard to determine the amount of a specific SSU rDNA sequence.

scholarly journals Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

2009 ◽  
Vol 29 (6) ◽  
pp. 469-473 ◽  
Author(s):  
Edna Maria Cavallini Sanches ◽  
Susi M. Pacheco ◽  
Alison S. Cericatto ◽  
Rosane M. Melo ◽  
Edson Molleta Colodel ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Urban Areas ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Rrna Gene ◽  
Wild Animals ◽  
Tadarida Brasiliensis ◽  
Desmodus Rotundus ◽  
Mato Grosso ◽  
Wide Range

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Relationships among the three major lineages of the Acari (Arthropoda:Arachnida) inferred from small subunit rRNA: paraphyly of the Parasitiformes with respect to the Opilioacariformes and relative rates of nucleotide substitution

2005 ◽  
Vol 19 (5) ◽  
pp. 383 ◽  
Author(s):  
Anna Murrell ◽  
Susan J. Dobson ◽  
David E. Walter ◽  
Nick J. H. Campbell ◽  
Renfu Shao ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Relative Rate ◽  
Sister Group ◽  
Small Subunit ◽  
High Rate ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Nucleotide Substitutions ◽  
Generation Times ◽  
Nucleotide Substitution Rates

We inferred phylogeny among the three major lineages of the Acari (mites) from the small subunit rRNA gene. Our phylogeny indicates that the Opilioacariformes is the sister-group to the Ixodida+Holothyrida, not the Ixodida+Mesostigmata+Holothyrida, as previously thought. Support for this relationship increased when sites with the highest rates of nucleotide substitution, and thus the greatest potential for saturation with nucleotide substitutions, were removed. Indeed, the increase in support (and resolution) was despite a 70% reduction in the number of parsimony-informative sites from 408 to 115. This shows that rather than ‘noisy’ sites having no impact on resolution of deep branches, ‘noisy’ sites have the potential to obscure phylogenetic relationships. The arrangement, Ixodida+Holothyrida+Opilioacariformes, however, may be an artefact of long-branch attraction since relative-rate tests showed that the Mesostigmata have significantly faster rates of nucleotide substitution than other parasitiform mites. Thus, the fast rates of nucleotide substitution of the Mesostigmata might have caused the Mesostigmata to be attracted to the outgroup in our trees. We tested the hypothesis that the high rate of nucleotide substitution in some mites was related to their short generation times. The Acari species that have high nucleotide substitution rates usually have short generation times; these mites also tend to be more active and thus have higher metabolic rates than other mites. Therefore, more than one factor may affect the rate of nucleotide substitution in these mites.

scholarly journals Two new brown rot polypores from tropical China

MycoKeys ◽  
2021 ◽  
Vol 82 ◽  
pp. 173-197
Author(s):  
Meng Zhou ◽  
Chao-Ge Wang ◽  
Ying-Da Wu ◽  
Shun Liu ◽  
Yuan Yuan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Southern China ◽  
Brown Rot ◽  
Small Subunit ◽  
Sensu Stricto ◽  
Wood Products ◽  
Rrna Gene ◽  
Ribosomal Rna Gene

Brown-rot fungi are types of fungi that selectively degrade cellulose and hemicellulose from wood and are perhaps the most important agents involved in the degradation of wood products and dead wood in forest ecosystem. Two new brown-rot species, collected from southern China, are nested within the clades of Fomitopsis sensu stricto and Oligoporus sensu stricto, respectively. Their positions are strongly supported in the Maximum Likelihood phylogenetic tree of the concatenated the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nuSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-α gene (TEF1) sequences. Fomitopsis bambusae, only found on bamboo, is characterised by its resupinate to effused-reflexed or pileate basidiocarps, small pores (6–9 per mm), the absence of cystidia, short cylindrical to oblong-ellipsoid basidiospores measuring 4.2–6.1 × 2–2.3 μm. Oligoporus podocarpi is characterised by white to pale cream pore surface, round or sometimes angular pores (5–6 per mm), broadly ellipsoid to reniform basidiospores measuring 3.8–4.2 × 2–2.3 μm and growing on Podocarpus. Illustrated descriptions of these two novel species, Fomitopsis bambusae and Oligoporus podocarpi, are provided.

scholarly journals Prevalence and Genetic Characterization of Cryptosporidium Infection in Java Sparrows (Lonchura oryzivora) in Northern China

2017 ◽  
Vol 2017 ◽  
pp. 1-4 ◽  
Author(s):  
Qiu-Xia Yao ◽  
Xiao-Xuan Zhang ◽  
Kai Chen ◽  
Jian-Gang Ma ◽  
Wen-Bin Zheng ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Northern China ◽  
Small Subunit ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Wide Range ◽  
Baseline Information ◽  
And Control

Cryptosporidiosis is a cosmopolitan parasitosis that affects a wide range of hosts including birds. As information concerning Cryptosporidium in birds is limited, the present study examined the prevalence and genotypes of Cryptosporidium in Java sparrows in Beijing and Shangqiu, northern China. Three hundred and fifty fecal samples were collected from Java sparrows (Lonchura oryzivora, 225 white Java sparrows and 125 gray Java sparrows) in Beijing and Shangqiu in October 2015, and the samples were examined by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. The overall Cryptosporidium prevalence is 13.42% (47/350), with 16.44% (37/225) in white Java sparrows and 8.00% (10/125) in gray Java sparrows. Cryptosporidium prevalence was 9.82% (16/163) in Java sparrows from Beijing and 16.58% (31/187) in Java sparrows from Shangqiu. The prevalence of Cryptosporidium in females and males was 40.63% (26/64) and 7.34% (21/286), respectively. The Cryptosporidium prevalence in Java sparrows of different ages varied from 10.47% to 16.33%. Sequence analysis of the SSU rRNA gene revealed that all the samples represented C. baileyi. This is the first report of Cryptosporidium in gray Java sparrows in China, which extend the host range for C. baileyi. These results provide baseline information for further studies of molecular epidemiology and control of Cryptosporidium infection in poultry in China.

Morphological reports on two species of Dexiotricha (Ciliophora, Scuticociliatia), with a note on the phylogenetic position of the genus

2014 ◽  
Vol 64 (Pt_2) ◽  
pp. 680-688 ◽  
Author(s):  
Xinpeng Fan ◽  
Saleh A. Al-Farraj ◽  
Feng Gao ◽  
Fukang Gu
Keyword(s):  
Phylogenetic Analyses ◽  
Eastern China ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Contractile Vacuole ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
North West ◽  
Rdna Sequences

Two Dexiotricha species (Dexiotricha elliptica nov. comb. and Dexiotricha cf. granulosa), respectively isolated from soil north-west of Riyadh, Saudi Arabia, and freshwater in Shanghai, eastern China, were investigated using standard methods. The species Loxocephalus ellipticus Kahl, 1931 is reclassified here in the genus Dexiotricha and was characterized mainly by constantly showing 16 somatic kineties, three post-oral kineties with the middle one shortened, a contractile vacuole located subcaudally with an excretory pore near the posterior end of somatic kinety 2 and single caudal cilia. A Dexiotricha granulosa-like organism having a subcaudally located contractile vacuole and fewer somatic kineties was designated D. cf. granulosa. The small-subunit rRNA gene (SSU rDNA) sequences of these two species were characterized and their phylogenetic positions based on SSU rDNA sequences were revealed by means of Bayesian inference and maximum-likelihood analysis. Phylogenetic analyses confirmed Dexiotricha as a monophyletic genus and supported its assignment to the order Loxocephalida. However, its family assignment remains unsupported.

scholarly journals Molecular identification and subtyping of Blastocystis sp. in laboratory rats in China

Parasite ◽  
2020 ◽  
Vol 27 ◽  
pp. 35
Author(s):  
Junqiang Li ◽  
Yueyue Yuan ◽  
Yuxi Jiang ◽  
Wen Wang ◽  
Liqin Chao ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Small Subunit ◽  
Laboratory Animals ◽  
Rrna Gene ◽  
Sprague Dawley ◽  
Laboratory Rearing ◽  
Spontaneously Hypertensive ◽  
Laboratory Rats ◽  
Oral Transmission ◽  
Blastocystis Sp

Blastocystis sp. is a ubiquitous protist that has been frequently reported in humans and animals worldwide. A total of 355 fecal samples of experimental rats were collected from four laboratory rearing facilities in China, and Blastocystis sp. was detected by PCR amplification of the partial small subunit ribosomal (SSU) rRNA gene. Twenty-nine (8.2%, 29/355) samples were positive for Blastocystis sp., with the highest infection rate (20.7%, 24/116) in rats of the Zhengzhou1, followed by that in the Zhengzhou2 (5.0%, 2/40), Shenyang (3.0%, 3/100) and Wuhan (0) rearing facilities. Among the three rat strains, Sprague–Dawley (SD) rats had higher infection rates (11.3%, 17/151) compared to Wistar rats (8.7%, 9/104) and spontaneously hypertensive (SH) rats (3.0%, 3/100). Two Blastocystis sp. subtypes (ST4 and ST7) were identified. ST4 was the predominant subtype detected in 26 samples (89.7%). A phylogenetic analysis demonstrated that the sequences of ST4 and ST7 obtained in this study were clustered with their reference subtypes. To our knowledge, this is the first report of Blastocystis sp. in experimental rats in China. Pathogen infections in laboratory animals need to be monitored due to fecal-oral transmission.

scholarly journals Nakazawaea Odontotermitis f.a., sp. nov., a Novel Yeast Isolated from the Gut of Odontotermes Horni in India.

2021 ◽  
Author(s):  
Snigdha Tiwari ◽  
Bhaskar C. Behera ◽  
Abhishek Baghela
Keyword(s):  
New Species ◽  
Type Strain ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Its Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
Ascomycetous Yeast ◽  
Lsu Rrna Gene

Abstract Three strains SMT1.3, SMT1.10, and SMT2.2, representing a novel asexual ascomycetous yeast species, were isolated from the gut of a termite Odontotermes horni in Maharashtra, India. Phylogenetic analyses of the LSU, ITS and SSU sequences revealed that they belonged to the genus Nakazawaea, with N. siamensis as the closest relative. The new species differed from the type strain of N. siamensis (DMKU-RK467T) by 1.93% nucleotide substitutions in the D1/D2 region of the large subunit (LSU) rRNA gene, 0.53% nucleotide substitutions in the small subunit (SSU) rRNA gene and 12.6% nucleotide substitutions in the internal transcribed spacer (ITS) region. Notable physiological differences were also observed between N. siamensis and the new species. Hence, the species Nakazawaea odontotermitis f.a., sp. nov. is proposed. The type strain is SMT1.3T (MTCC 13105 = NFCCI 5011). The GenBank accession numbers of the LSU and ITS and SSU sequences of Nakazawaea odontotermitis f.a., sp. nov. are MZ234240, MZ234239 and OK384663. The MycoBank number is MB 841926.

scholarly journals Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

1998 ◽  
Vol 64 (11) ◽  
pp. 4522-4529 ◽  
Author(s):  
Marcelino Suzuki ◽  
Michael S. Rappé ◽  
Stephen J. Giovannoni
Keyword(s):  
Clone Library ◽  
Fluorescence Emission ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Length Variation ◽  
Small Subunit Rrna ◽  
Gene Frequencies ◽  
Rdna Sequences

ABSTRACT Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

scholarly journals Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolyticaInfection

1998 ◽  
Vol 36 (2) ◽  
pp. 449-452 ◽  
Author(s):  
Rashidul Haque ◽  
I. K. M. Ali ◽  
S. Akther ◽  
William A. Petri
Keyword(s):  
Specific Antigen ◽  
Small Subunit ◽  
Antigen Detection ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Isoenzyme Analysis ◽  
Pcr Product ◽  
Stool Samples ◽  
Stool Specimens ◽  
Fresh Stool

The diagnosis of amebiasis by microscopic identification of the parasite in stool is insensitive and unable to distinguish the invasive parasite Entamoeba histolytica from the commensal parasite E. dispar. In this study, we have tested a PCR technique for the detection of E. histolytica and compared it with isoenzyme analysis and the TechLab E. histolytica-specific antigen detection test. The nested-PCR test we used is based on amplification of the small subunit rRNA gene ofE. histolytica and E. dispar followed by restriction digest analysis of the PCR product. Single stool samples were obtained from 98 patients from Dhaka, Bangladesh, with diarrhea: 88 patients diagnosed by microscopy and/or culture with E. histolytica and/or E. dispar infection and 10 patients without infection. Isoenzyme analysis identified 53 of the infections as E. histolytica and 28 as E. dispar. PCR and isoenzyme identification of E. histolytica agreed in 96% (51 of 53) of amebic cultures. PCR forE. histolytica was negative in all 10 samples that were negative for E. histolytica by isoenzyme and antigen detection. PCR and antigen detection had comparable sensitivities when performed directly on fresh stool specimens, identifying 87% (46 of 53) and 85% (45 of 53), respectively, of E. histolyticainfections identified by isoenzyme analysis. The correlation of results by antigen detection and PCR for identification of E. histolytica in stool was 93% (45 of 48 cases). Mixed infections with E. histolytica and E. dispar were detected by PCR in 14% (12 of 88) of cases. In conclusion, all three techniques for specific identification of E. histolytica in fresh stool showed excellent correlation. Only the TechLab E. histolytica antigen detection test was both rapid and technically simple.

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