Restriction Enzymes HaeIII Research Articles

Application of nested PCR in P1-RFLP for genotyping Mycoplasma pneumoniae strains

Objective To evaluate the efficiency of using nested PCR in restriction fragment length polymorphism analysis (P1-RFLP) for genotyping Mycoplasma pneumonia (M.pneumonia) in clinical specimens.Methods Based on the gene sequence of RepMp4 and RepMp2/3 in P1 gene of reference strains M129 (type 1) and FH (type 2),two sets of inner primers were designed with a HaeⅢ restriction enzyme site (GGCC).The nested PCR was set up to detect the target DNA in clinical specimens.The amplification products were mixed and digested with Hae Ⅲ enzyme.The genotypes were analyzed by comparing with various restriction maps and the results were verified by sequencing analysis.The concentration of DNA extracted from standard and clinical strains were detected by ten-fold dilution to evaluate the sensitivity of nested PCR-P1-RFLP and P1-RFLP.M.pneumonia-positive specimens isolated from Beijing in 2012 were analyzed by the nested PCR-P1-RFLP and the results were compared with those by P1-RFLP analysis.Results The nested PCR-P1-RFLP could effectively genotype M.pneumonia in clinical specimens and the results were consistent with those by sequencing analysis.The sensitivity of new assay was 103 times higher than that of the original P1-RFLP.Of the 115 M.pneumoniae positive clinical specimens,97.4％ (112/115) were type 1 and the rest were type 2.Conclusion The nested PCR-P1-RFLP shows high efficiency for genotyping of M.pneumonia in clinical specimens.It might be useful for the surveillance of M.pneumoniae infection. Key words: Mycoplasma pneumoniae; Genotyping; Nested-PCR; P1-RFLP

An Improved Protocol for Extraction of Metagenomic DNA from High Humus, Alkaline and Saline Soil of Chinampa for T-RFLP Fingerprinting Analysis

Aims: The principle aim of this study was to obtain high quality metagenomic DNA from the high humus-containing, alkaline soils of the chinampas, an artificial sustainable agroecosystem. Study Design: Different protocols reported previously were tested and were modified to extract the metagenomic DNA. Quality of the DNA samples was evaluated by amplification of 16S rRNA gene with PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis. Place and Duration of Study: This study was performed in Department of Microbiology, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional during 2011-2012. Methodology: Four soil samples were collected from two chinampas at the depth of 0-30 Original Research Article British Microbiology Research Journal, 4(7): 821-830, 2014 822 cm and 30-60cm. A protocol started with repeated prewashing before the direct cell lysis with lysozyme followed by SDS treatments, frozen and melting cycling was developed which combined the DNA isolation and purification procedures. The 16SrRNA genes were amplified from the extracted metagenomic DNAs and were used for T-RFLP fingerprinting analysis. Results: The 16SrRNA genes were amplified from all the DNA extracts corresponding to the four soil samples and were successfully used in the T-RFLP analysis, which generated 25 to 109T-RFs in the four soil samples digested separately with the restriction endonucleases HaeIII, HhaI and MspI. Conclusion: The protocol developed in the present study could generate high molecular weight and high quality metagenomic DNA from soils with high content of humic materials, for which the other reported protocols were not functioned. This soil harboured very diverse and unique bacterial communities belonging to at least nine phyla that might contribute to the high soil fertility.

Analysis of the Human Oral Microbiome of Smokers and Non-Smokers Using PCR-RFLP and Ribotyping

Recent advances in the field of microbial and medical ecology emphasize the critical role played by oral bacteria in the delicate dynamic equilibrium of human health and disease, creating the need to define the bacterial communities associated with healthy and non-healthy conditions and to capture shifts in community structure germane to diagnosis. Employing PCR-RFLP of the 16S rDNA gene from metagenomes and plate-wash (cultured) bacteria of oral wash from 10 volunteers, this study evaluated the stability of oral bacteria in healthy subjects and documented community shifts in smokers. Sequence analysis of selected 16S gene amplicons cloned with the Gene Hunter PCR-Trap vector and pCR 4-TOPO cloning kits was conducted to determine the bacteria identity and diversity indices of the two groups. Ribopatterns generated by the restriction enzymes HaeIII and Sau3AI were significantly (p AluI using the GelCompare II software cluster analysis. A stable core of bacteria DNA fingerprint was detected in all healthy subjects, and remained unchanged over the study period of 3 months. Signature bands (1500 bp with HaeIII) in smokers and in non-smokers (800 bp and 700 bp with Sau3A1) were evidently suggesting the presence of potential biomarkers of healthy and non-healthy states. There was no significant difference in the DNA fingerprints of cultured and metagenomic extracts. The genera Xanthomonas, Streptococcus and phylum Candidatus occurred in large numbers in both groups, however, a major shift in composition with the dominance of gram-negative bacteria in smokers compared to healthy subjects was quite remarkable. Taxonomic diversity in smokers was quite high, including members of the genera Rothia, Synechococcus, Neisseria, Thiomargarita and Pyrobaculum. These data highlight the presence of a stable core microbiome amidst a wide diversity, identify a distinct smokers’ cluster and open the way for the search for potential biomarkers for specific diseases.

An Electrochemical Assay for Restriction Endonuclease Activity Using Graphene Monolayer

Herein, we report a new electrochemical assay for the endonuclease activity using a graphene monolayer. The approach involves a methylene blue (MB) tagged double-stranded (ds) DNA as an enzyme substrate with a single-stranded (ss) segment at the end for anchoring on graphene through π-π stacking. The nuclease enzyme mediates DNA hydrolysis following the release of dsDNA-MB from the graphene surface, which reduces the electrochemical current signal. The protocol was validated for the HaeIII and EcoRV restriction endonuclease enzyme. We also assayed the activity of methyltransferase enzyme and inhibition of restriction by HaeIII. The method allows real-time measurement and quantitative assay for endonuclease activities in a simple manner.

Molecular characterisation of T cell receptor-zeta subunit (CD247) gene in buffalo (Bubalus bubalis)

The cDNA encoding T cell receptor-zeta (TCR-ζ; CD247) molecule of water buffalo (Bubalus bubalis) was isolated, cloned and sequenced in the present study. The CD247 cDNA comprised 1078 nucleotides including a 30 nucleotide 5′-untranslated region (UTR), 495 nucleotide single open reading frame (ORF) and 553 nucleotide 3′-UTR. Deduced amino acid of buffalo CD247 sequence was two residues shorter than the corresponding cattle and sheep sequences. However, ruminant-specific insertions and substitutions in intra-cytoplasmic (IC) domain were present in buffalo. Immunoreceptor tyrosine-based activation motifs (ITAMs), the important motifs for TCR signalling, were totally conserved among ruminants including buffalo. The 3′-UTR region of the buffalo CD247 was highly homologous to the corresponding region in the cattle sequence and showed lack of polymorphism after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using HaeIII and MseI restriction enzymes in buffalo population. Phylogenetically, buffalo sequence was closer to cattle sequence under the ruminant's lineage. The conserved nature of this gene ensures TCR integrity which is vital for induction of optimal and efficient immune response.

Development of Assays Using Hexokinase and Phosphoglucomutase Gene Sequences That Distinguish Strains of Leishmania tropica from Different Zymodemes and Microsatellite Clusters and Their Application to Palestinian Foci of Cutaneous Leishmaniasis

Background/ObjectivesPalestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307.Methodology/Principle FindingsAssays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system ‘correctly’ identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter.Conclusions/SignificanceBoth assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.

Detection of Human Cytomegalovirus UL97 D605E Mutation in Korean Stem Cell Transplantation Recipients and Donors

Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCRbased restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.

Genotypic diversity in native rhizobial population nodulating Vicia faba in arid and semi-arid regions of Haryana state (India)

A Rhizobium–legume interaction stands out from other plant–microbe interactions as one in which a true developmental mutualism occurs. To study the genotypic diversity in native population of rhizobia-nodulating Vicia faba plants, we retrieved 64 rhizobial isolates from root nodules of faba bean grown in pots holding soils collected from arid and semi-arid regions of the state of Haryana, India. The amplification of nodC in all the isolates authenticated these as rhizobia. The nitrogen-fixing potential of the isolates was tested by the amplification of the nifH gene. Only 50 isolates out of 64 showed nifH gene amplification. The characterization of the isolates by amplified 16S rDNA restriction analysis (ARDRA) categorized these into 36 16S rDNA genotypes using a combination of MspI and HaeIII restriction enzymes. Majority of the isolates resolved into separate genotypes, indicating a wide diversity among them, which seemed to arise from their geographical origin and soil characteristics. These findings may be immensely useful in agriculture towards developing rhizobial inoculants specific for faba beans under arid and semi-arid conditions.

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB.

First report of the association of a ‘Candidatus Phytoplasma ulmi’ isolate with a witches' broom disease of reed in China

Phragmites australis (Poaceae) is widely distributed around the world. It is not only a perennial weed, but also a raw material for the paper industry, a medicinal plant, animal fodder, an ornamental in landscaping and a building material. It now covers approximately two million ha in China, including ten thousand ha in Xinjiang Province. During September 2012, symptoms of witches' broom, stunting, and smaller leaves (Fig. 1A) were observed in over 57% of P. australis plants at the Shihezi University Experimental Station (Shihezi, Xinjiang Province). Leaf samples were collected from both symptomless plants and plants showing symptoms. Total DNA was extracted from 0.5 g fresh leaf midribs from P. australis plants with and without symptoms, using the CTAB method (Angelini et al., 1). The DNA extracts were tested for phytoplasma infection by a nested polymerase chain reaction (PCR) assay with phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 2) and R16F2n/R16R2 (Gundersen & Lee, 3). The two primer pairs produced PCR products of 1.8 and 1.2 kb, respectively, from most of the plants with symptoms (4/5), but not from the symptomless plants (2/2). R16F2n/R16R2 amplicons were cloned (pMD18-T simple vector, TaKaRa, China) and sequenced. All of the phytoplasma R16F2n/R16R2 sequences were 100% identical to each other. BLAST comparisons showed that the P. australis witches' broom phytoplasma 16S rDNA consensus sequence (GenBank Accession No. KC331052) had 99% sequence identity with those of members of group 16SrV (‘Candidatus Phytoplasma ulmi’). Similarity coefficients and in silico restriction fragment length polymorphism (RFLP) analyses of the R16F2n/R16R2 sequence with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, I, and TaqI restriction endonucleases (iPhyClassifier, http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) (Fig. 2), indicated that P. australis contained a phytoplasma strain identical to those of the reference strain AB052876, ‘Ca. Phytoplasma ulmi’, subgroup 16SrV-B. A neighbour-joining dendrogram (MEGA 5.0, USA) based on the 16S rDNA sequences of the P. australis witches' broom phytoplasma and twenty-five reference phytoplasmas in NCBI database supported the PCR and RFLP results since the P. australis witches' broom phytoplasma grouped within the 16SrV-B phytoplasma clade (Fig. 3). The group 16SrV was previously associated with witches' broom diseases of several plant species in China including jujube (Zhu et al., 6), paper mulberry (Liu et al., 4), and Chinese honeylocust (Min et al., 5). This is the first report of a ‘Ca. Phytoplasma ulmi‘-related strain affecting P. australis in China. This research was supported by the National Key Technology R&D Program of China (2011BAD48B00).

Novel molecular marker associated with Tm2a gene conferring resistance to tomato mosaic virus in tomato

AbstractTomato mosaic virus (ToMV) is an important Tobamovirus that causes significant crop losses. Resistance to the ToMV is conferred by the genes Tm1, Tm2 and Tm2a. Among these three genes, Tm2a confers resistance to most strains of the ToMV. Screening of genetic lines under field conditions based on phenotype is time‐consuming and challenging due to concerns associated with stability of the virus and its potential transmission to other plants. Tightly linked molecular markers associated with resistance genes can improve selection efficiency and avoid these problems. This study developed a PCR‐based marker based on restriction site differences from Tm2a locus‐specific sequences, which was found to be useful in identifying the resistant and susceptible genotypes and was consistent with phenotypic data. The marker is a codominant cleaved amplified polymorphic sequence (CAPS) marker producing 270‐ and 600‐bp DNA fragments from resistant genotypes and an 870‐bp fragment from susceptible genotypes when digested with HaeIII restriction enzyme. This novel marker can be useful for tomato breeders to screen progeny from segregating populations for ToMV resistance.

English

Traditional methods that are used to identify clinical isolates of&nbsp;Candida&nbsp;species are time-consuming and not appropriate for rapid, accurate and reliable identification. The aim of this study was to characterize&nbsp;Candida&nbsp;species isolated from fingernail onychomycosis by using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) with three different restriction enzymes HaeIII, DdeI and BfaI. 16Candida&nbsp;isolates recovered from infected fingernail samples were selected and phenotypically identified by API 20C identification kit. Also, the five species were identified genotypically with PCR-RFLP analysis. 16 clinical&nbsp;Candida&nbsp;isolates comprising five species were included in this study; three&nbsp;Candida&nbsp;species were identified using conventional API 20C:&nbsp;C. albicans&nbsp;(9/16),&nbsp;C. parapsilosis&nbsp;(3/16) and&nbsp;C. glabrata&nbsp;(1/16) with three unidentified isolates. Five species were identified based on genotypic identification with PCR-RFLP analysis revealing&nbsp;C. albicans (9/16), C. parapsilosis&nbsp;(4/16),C. glabrata,&nbsp;C. guilliermondii&nbsp;and&nbsp;C. tropicalis&nbsp;(1/16 for each). It was concluded that the PCR-RFLP analysis of rDNA enabled more easy, rapid and precise identification ofCandida&nbsp;species compared to phenotypic methods. RFLP analysis demonstrated that HaeIII is the most differentiating enzyme that distinguished&nbsp;C. albicans&nbsp;from non&nbsp;C. albicans&nbsp;species. BfaI maintained the differentiation of these non&nbsp;C. albicans&nbsp;species and DdeI digestion confirmed&nbsp;C. albicans&nbsp;species. &nbsp; Key words:&nbsp;Onychomycosis,&nbsp;Candida&nbsp;species, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

PCR-RFLP analysis of fliC, fimH and 16S rRNA genes in Salmonella Typhimurium isolates of varied origin

Restricted fragment length polymorphism (RFLP) was used in analyses on the typing and heterogeneity, typeability and polymorphism of the 16S rRNA, fliC and fimH genes in Salmonella Typhimurium isolates of varied origin. The digestion of PCR products with restriction enzymes EcoRV, ClaI, HaeIII and ScaI (fliC genes), HincII, ClaI, EcoRV and MluI (fimH genes) and EcoRI, SmaI and HaeIII (16S rRNA genes) generated two to four bands of ranging in size from 100 to 1,104 bp. Of all the restriction profiles obtained, only the ClaI profile for fimH could be used to classify Salmonella Typhimurium isolates into different groups. According to this profile, pattern A with uncut fimH was observed in eight isolates (36.36 %) and pattern B with 755- and 253-bp bands was observed in 14 isolates (63.63 %). No pattern was allotted for a special region or source. These results demonstrate that PCR-RFLP based on these genes showed good typeability but low discriminatory power. Moreover, the highly conserved nature of fliC, fimH and 16S rRNA illustrated in our study suggests the importance of these genes as immunization and diagnostic factors in Salmonella Typhimurium. Simultaneously, our results also illustrate the potential of ClaI-based fimH analysis as a marker for the sub-serotype level differentiation of Salmonella Typhimurium isolates.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Using PCR-RFLP for sexing of the endangered Galápagos petrel (Pterodroma phaeopygia)

Pterodroma phaeopygia is a critically endangered avian species of the Galápagos Islands. This bird is sexually monomorphic, making it difficult to identify the sex. This information, however, is relevant to studies of behavior, ecology, and management of wild or captive populations. Here, we aimed to implement a molecular approach for determining sex in this petrel. DNA was extracted from the blood and the feathers of 24 adult P. phaeopygia, with samples from a female and a male Gallus gallus for comparison. We amplified the cromo-helicase DNA binding protein 1 (CHD-1) gene by PCR, using primers P2 and P8. Allele CHD-1W is unique to females and CHD-1Z occurs in both sexes. We then digested these PCR products using the restriction enzyme HaeIII. The PCR amplified a 400-bp product for both alleles. The digestion of the G. gallus, amplicons split the CHD-1Z allele into two fragments (of 320 and 80 bp), while CHD-1W remained intact. Thus, the male exhibited two bands (digested CHD- 1Z) and the female three bands (undigested CHD-1W and digested CHD-1Z). Applying this RFLP method on DNA derived from blood, 9 of the 24 petrels were found to be male, while 15 were females. The same results were obtained using feathers as the source of DNA. To our knowledge, this is the first report of molecular method for sexing this species. The potential of sexing this petrel from feathers is remarkable as it minimizes blood sampling induced stress. This method could be used to reinforce the conservation efforts for this bird, to investigate population sex ratios and to develop new conservation strategies.

Evaluation of Several DNA Extraction Methods for Obtaining Total Community DNA from Anaerobic Digestion Sludge

In order to develop and establish an effective method for extraction of total genomic DNA from anaerobic digestion sludge, the DNA extraction methods consisted of enzymatic-disruption method, physical–chemical method, and Mp- Bio method were used to extract DNA from anaerobic digestion sludge samples and compared with in terms of the efficient cell lysis, DNA yield, purity and molecular analyses. The results showed that total genome DNA could be extracted from anaerobic digestion sludge by all the three methods. However, the enzymatic method had better DNA yields, cell lysis efficiency and lower humic acids and expensive than other DNA extraction methods. Total DNA was purified by spin-bind cartridge after being precipitated by PEG8000, the humic acids content was reduced by 96% during the purification steps, and the DNA recovery efficiencies were high and similar each other, which were about 88%. A eubacterial 16S rRNA gene-targeted primer pair was used for PCR polymerase chain reaction to amplify a region of the 16S rRNA to check the purity of the purified sample. Results indicated that nearly full length 16S rDNAs were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzymes HhaI and HaeIII. All of these above indicated that the enzymatic-disruption method is the best method to obtained genome DNA from anaerobic digestion sludge.

Genetic Selection Barriers in Global Development of Rural Goat Production and a Simplified Approach in Identification of Proper Polymorphic Types

The lack of enough funding for rural development projects is hindering the genetic characterization of goats around the globe. This is the first simplified recognition of multiple polymorphism of production, immunity, and health genes of sperms in rural goats, paving in its potential towards adoption of this model for future selection programs, targeting a direct improvement of rural goat herds, and aiming at an indirect benefit to the poor shepherd’s livelihood around the globe. The multiple polymorphism was studied in the sperms’ production genes (Growth Hormone, and αS1-Casein), immunity gene (MHC Class II DRB), and Transmissible Spongiform Encephalopathy (TSE) resistant gene (PrP). The known genome of Saanen goat sperms was used as a control-reference in this research. There was a difference in the frequency of polymorphic types in PCR-amplicons of Growth Hormone subjected to HaeIII restriction endonuclease, of αS1-Casein subjected to XmnI enzyme, MHC class II DRB subjected to TaqI endonuclease, and PrP subjected to amino acid sequencing. The recognition of multiple polymorphisms in important production, immunity, and health related sperm genes of rural goats, could be used in a reliable model for establishing future selection programs aiming at improvement of food security and safety in rural areas.

Isolation and Characterization of Enterococci Bacteriocinic Strains from Tunisian Fish Viscera

A collection of lactic acid bacteria isolates from fish viscera was studied and investigated regarding to their functional properties and safety aspects. From these, three isolates GM1, GM2 and GM3 were identified as Enterococcus feacium species using molecular methods. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with restriction enzyme HaeIII separated these isolates into distinctive group which suggest genotypic variability within enterococci strains isolated from fish viscera. The three strains GM1, GM2 and GM3 exhibited antimicrobial activity. Indeed strains have been shown to produce bacteriocins with inhibitory effect against food spoilage bacteria and pathogenic fish including Carnobacterium maltaromaticum. The molecular mass of bacteriocin, as calculated by tricine-SDS-PAGE, was found to be 4.5 kDa. All isolates were tested positive upon PCR amplification of enterocin A structural gene. Investigations of antibiotic resistance show that the isolates were mostly sensitive to several antibiotics (ampicillin, penicillin, tetracycline, gentamycine) and resistance to rifampicin. All isolates grow in esculin azide agar as a selective medium for enumeration of probiotic enterococci. This study suggests that our strains can be employed as probiotic or to improve the safety of food products.

제주도에서 채집한 해양 해면, Asteropus simplex의 공생세균에 관한 계통학적 분석

해양 해면 Asteropus simplex를 제주도에서 채집하여 배양에 의한 RFLP와 비배양에 의한 DGGE 분석 방법에 의해 세균군집 구조를 조사하였다. 16S rDNA-RFLP 분석을 위해 변형된 Zobell 배지와 MA를 이용하여 120균주를 선별하고 제한효소, HaeIII와 MspI을 사용하여 각각의 다른 RFLP 패턴으로 구분하였다. RFLP 패턴으로부터 유래한 16S rDNA 염기서열 분석결과, 알려진 세균 종과 94% 이상의 유사도를 나타내었으며 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, 5개의 문이 관찰되었다. 그 중 Gammaproteobacteria가 우점하였다. 같은 해면, A. simplex의 DGGE 분석을 위해 total genomic DNA로부터 16S rDNA를 증폭하여 DGGE fingerprinting을 수행한 결과 12개의 서로 다른 밴드가 관찰되었다. 각 밴드의 16S rDNA 염기서열은 알려진 세균의 염기서열과 90% 이상의 유사성을 나타내었으며 대부분의 염기서열은 uncultured bacteria에 속하였다. DGGE 분석으로부터 미생물의 군집은 Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Chloroflexi, Nitrospira, 7개의 문으로 나타났다. RFLP와 DGGE 방법에 의해 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria가 공통적으로 발견되었으나 전체적인 공생세균의 군집구조는 분석방법에 따른 차이를 나타내었다. 배양에 의한 방법보다 비배양 방법에서 더 다양한 세균군집구조를 나타내었다. Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Asteropus simplex collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and MA media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 94% similarities compared with known bacterial species, and the isolates belonged to five phyla, Alphaproteobacteria, Gammaproteobacteria Actinobacteria, Bacteroidetes, and Firmicutes, of which Gammaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge-derived total gDNA showed 12 DGGE bands, and their sequences showed more than 90% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven phyla, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteira, Chloroflexi, and Nitrospira. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with A. simplex by both RFLP and DGGE methods, however, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture-independent method than in culture-dependent method.

PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data

BackgroundIn molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems.ResultsPyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10′000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes.ConclusionsPyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run.