Restriction analysis of an amplified polygalacturonase gene fragment differentiates strains of the phytopathogenic bacterium Pseudomonas solanacearum

Amplification of a polygalacturonase gene fragment using the polymerase chain reaction (PCR) formed a rapid, sensitive and portable method for detecting and differentiating strains of Pseudomonas solanacearum , a taxonomically complex bacterial species. Primers 5'CAG CAG AAC CCG CGC CTG ATC CAG 3' and 5'ATC GGA CTT GAT GCG CAG GCC GTT 3' were used to amplify a 504 base pair polygalacturonase gene fragment from 57 Ps. solanacearum isolates. Digestion of these products with Hae III defined groups which reflected the known genetic divisions within the species.     

PCR Amplification of Tetrahymena rDNA Segments Starting with Individual Cells

To facilitate studies of rDNA molecular genetics in Tetrahymena thermophila , we attempted the detection of polymorphisms in the nontranscribed spacers (NTSs) using polymerase chain reaction (PCR), starting with minute amounts of DNA. The targeted polymorphic regions are 85% adenine-thymine (AT). We found conditions of efficient and specific in vitro amplification of targeted segments in the replication domain of the 5'NTS and in the subtelomeric segment of the 3'NTS. The identity of the amplified segments was confirmed by restriction enzyme digestion and DNA sequence analysis. Digestion of the template DNA at restriction sites upstream and downstream of the targeted region increased the efficiency of amplification, presumably because the targeted segments are in a palindromic molecule. Starting from total cell DNA corresponding to as little as 0.03 picogram (equivalent to the DNA content of 0.003 cells or about 30 rDNA molecules), we observed the amplified band after agarose gel electrophoresis and ethidium bromide staining. The yield indicated more than 10-billion-fold amplification. Amplification of the subtelomeric fragment yielded homogeneous product of minimum possible length even though the telomeric-specific primer can bind, at least initially, at a multiplicity of GGGGTT repeats. Amplified 5'NTS product also was detected in an ethidium-bromide-stained gel when PCR was started with a single cell.     

Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

The objective of this study was to determine the genetic relatedness among the Cercospora and Pseudocercospora species closely related to Cercospora apii by using a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the internal transcribed spacer (ITS) region. A single PCR fragment (about 550 bp) was obtained from all Cercospora species categorized as the C. apii-group, Pseudocercospora purpurea, Pseudocercospora conyzae, and Pseudocercospora cavarae. Cercospora caricis yielded a 680 bp PCR fragment. The similarity in the PCR fragment size and RFLP profiles among the C. apii-group isolates, including Pseudocercospora purpurea, and Pseudocercospora conyzae strongly suggests that these species are conspecific. Synonymy with C. apii (lectotype) at a subspecific rank has been proposed. Amplified ITS regions of genomic DNA extracted from spinach leaves showing 12 and 233% leaf spot disease symptoms caused by Cercospora beticola yielded two PCR fragments (i.e., one from the fungus and one from the host plant) and were resolved by electrophoresis of the PCR product in 3% LMP agarose. Digestion of the total PCR product with HinfI restriction enzyme yielded RFLP profiles similar to those obtained from amplified DNA from the causative agent, C. beticola. The method described in this preliminary study offers rapid detection and diagnosis of fungal infections in plants for disease prediction and management and screening of plant materials for quarantine purposes.     

Molecular epidemiology of Legionella pneumophilia serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting

Abstract Hybridization with acetylaminofluorene-labelled 16 + 23 S rRNA from Escherichia coli was used to detect DNA polymorphism among Legionella pneumophila serogroup 1 isolates. Isolates from unrelated patients showed at least four different rRNA restriction patterns, whereas those from related patients showed a single pattern. Amplification of genomic regions with an arbitrary primer by polymerase chain reaction was used to further analyze the isolates. Related isolates showed closely related patterns while unrelated isolates displayed six distinct patterns. We could differentiate the majority of unrelated isolates with the combination of the patterns obtained with the ribotyping and the PCR fingerprinting, while strains from the same outbreak remained highly related. The ribotyping and the PCR fingerprinting are proposed as useful and easy to perform epidemiological markers of L. pneumophila serogroup 1 infection.     

Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates

The genome of the Bacillus subtilis 168-type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI-H-G and rrnJ-W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168-type strain and B. subtilis 168-like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168-type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.     

PCR amplification of Tetrahymena rDNA segments starting with individual cells.

To facilitate studies of rDNA molecular genetics in Tetrahymena thermophila, we attempted the detection of polymorphisms in the nontranscribed spacers (NTSs) using polymerase chain reaction (PCR), starting with minute amounts of DNA. The targeted polymorphic regions are 85% adenine-thymine (AT). We found conditions of efficient and specific in vitro amplification of targeted segments in the replication domain of the 5'NTS and in the subtelomeric segment of the 3'NTS. The identity of the amplified segments was confirmed by restriction enzyme digestion and DNA sequence analysis. Digestion of the template DNA at restriction sites upstream and downstream of the targeted region increased the efficiency of amplification, presumably because the targeted segments are in a palindromic molecule. Starting from total cell DNA corresponding to as little as 0.03 picogram (equivalent to the DNA content of 0.003 cells or about 30 rDNA molecules), we observed the amplified band after agarose gel electrophoresis and ethidium bromide staining. The yield indicated more than 10-billion-fold amplification. Amplification of the subtelomeric fragment yielded homogeneous product of minimum possible length even though the telomeric-specific primer can bind, at least initially, at a multiplicity of GGGGTT repeats. Amplified 5'NTS product also was detected in an ethidium-bromide-stained gel when PCR was started with a single cell.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

apaH polymorphism in Clinical Actinobacillus actinomycetemcomitans isolates

In the present study the apaH polymorphism in clinical A. actinomycetemcomitans isolates were investigated in relation to their serotype and periodontal status of the donor subjects. The material included 122A. actinomycetemcomitans isolates representing serotypes a-e from 103 periodontally healthy and diseased subjects. The apaH polymorphism was investigated by both restriction analysis of the specific PCR amplification product and also by sequencing of PCR amplification products from selected clinical isolates. An apaH specific PCR amplification product was obtained from all isolates but the restriction patterns of the amplification products varied. Serotype c and genogroup 2 within serotype e formed genetically distinct groups, whereas isolates of serotype a, b, d and genogroup 1 within serotype e could not be separated from each other based on the apaH restriction analysis. No relation between the restriction pattern of apaH and the periodontal status of the individuals was detected. These results indicate that serotype c isolates form a uniformly distinct group within A. actinomycetemcomitans and that a subpopulation of serotype e isolates clearly diverge from all other A. actinomycetemcomitans isolates.     

Explorative multivariate analyses of 16S rRNA gene data from microbial communities in modified-atmosphere-packed salmon and coalfish

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.     

Polymorphisms in Phytophthora infestans: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions

Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of Phytophthora infestans. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with MspI uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with EcoRI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other Phytophthora species.     

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.     

Intersterility, morphology and taxonomy of Ceratocystis fimbriata on sweet potato, cacao and sycamore

Ceratocystis fimbriata is a large, diverse complex of species that cause wilt-type diseases of many economically important plants. Previous studies have shown that isolates in three monophyletic lineages within the Latin American clade of C. fimbriata are host-specialized to cacao (Theobroma cacao), sweet potato (Ipomoea batatas) and sycamore (Platanus spp.), respectively. We paired testers of opposite mating type from isolates of these lineages to find intersterility groups. Two intersterility groups corresponded to the sweet potato and sycamore lineages, respectively. The cacao lineage contained two intersterility groups, corresponding to two genetic sublineages centered in western Ecuador and Brazil/Costa Rica/Colombia. Six isolates from cacao that were not members of the cacao lineage and were not pathogenic to cacao in an earlier study also were intersterile with members of the two cacao intersterility groups. Some pairings between testers from different lineages or sublineages yielded perithecia from which a few abnormal progeny could be recovered, typical of interspecific hybrids. These progeny showed abnormal segregation of the MAT-2 gene and mycelial morphology, showing that they were indeed the result of crosses. Isolates of the sweet potato, cacao, and sycamore lineages were indistinguishable morphologically except for the presence or absence of a doliform (barrel-shaped) conidial state and minor differences in size of perithecial bases and necks and ascospores. C. fimbriata originally was described from sweet potato. We describe the cacao pathogen as a new species, Ceratocystis cacaofunesta and we raise the sycamore pathogen from a form to species Ceratocystis platani.     

Molecular cloning of PCR fragments with cohesive ends

Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids. Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized. The primers are chosen to span a known region of interest, and extended at their 5′-ends to include the desired restriction sites. Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers. A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s). Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize. Ligation products are used to transform competent bacteria. Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.     

Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests.

One hundred twenty isolates of Colletotrichum gloeosporioides from avocado (6 U.S. and 57 Israeli isolates) and almond (57 Israeli isolates) fruits were compared by various molecular methods and a pathogenicity assay in order to determine the genetic diversity and host specificity between and among the different populations. DNA from eight additional U.S. almond anthracnose isolates were also compared. PCR amplification of genomic DNA with four primers produced uniform banding patterns for all the Israeli almond isolates from different geographic locations in Israel. DNAs from the U.S. almond isolates were distinct from DNAs of the Israeli isolates. In contrast, the avocado isolates from Israel and the United States were more diverse, with numerous arbitrarily primed-PCR phenotypes being observed. HaeIII digestion patterns of A+T-rich DNA distinguished between the almond and avocado isolates. Southern hybridization of the repetitive nuclear-DNA element GcpR1 to PstI-digested genomic DNA of almond and avocado isolates revealed no polymorphic fragments among the almond isolates, whereas polymorphic fragments were observed among the avocado isolates. Amplification and subsequent restriction enzyme digestion of the internal transcribed spacer 4 and 5 regions between the small and large nuclear subunits of DNA encoding rRNA failed to distinguish between C. gloeosporioides isolates from a diverse host range. In artificial inoculations, avocado isolates produced various lesions on avocado and almond fruits, whereas the almond isolates infected both fruits at a lower rate.     

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.     

rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from S?o Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8 s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.     

Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the alpha-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten "atypical" E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two "atypical" E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Analysis of polymorphism of 18S rRNA gene in Wuchereria bancrofti microfilariae

The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes Ssp I, Msp I and Hha I showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any difference in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf isolates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.     

Explorative Multivariate Analyses of 16S rRNA Gene Data from Microbial Communities in Modified-Atmosphere-Packed Salmon and Coalfish

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.