Identification of Candida albicans by polymerase chain reaction amplification of CaYST1 gene intron fragment

A single pair of primers, deduced from the intron nucleotide sequence of the Candida albicans CaYST1 gene, was used in PCR analysis performed with both genomic DNA and whole cells of clinical isolates of Candida species and other microorganisms. All the clinical C. albicans isolates generated the expected 310 bp amplicon; other Candida species as well as laboratory strains belonging to other fungal genera failed to amplify any DNA fragment, except for Candida pseudotropicalis (amplicon of 1200 bp), Kluyveromices marxianus (amplicon of 1250 bp) and Cryptococcus neoformans (several amplicons longer than 1200 bp). Unusual C. albicans isolates from Africa also yielded the expected 310 bp amplicon. These results indicate that genes containing intron sequences may be useful to design species-specific primers for identification of fungal strains by PCR. The sensitivity of the method was evaluated for C. albicans genomic DNA by using both various DNA concentrations (224 ng to 2.7 pg) and different cell amounts (10(7); to 5 cells). The results obtained may be useful in earlier detection of candidiasis.     

Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.     

进口肉骨粉中牛成分检测研究

根据18S核糖体基因保守序列设计引物,对牛骨粉,鱼粉,牛肉,羊肉,猪肉,鸡肉,鸭肉,鱼肉的DNA样品进行PCR扩增,均得到137bp的扩增片段;根据牛线粒体的特异基因片段设计引物,对上述材料DNA样品进行PCR扩增,结果只在牛肉和牛骨粉中出现271bp的扩增,本实验建立了从进口肉骨粉中检测牛成分的PCR方法,检测灵敏度达到牛成分含量为0．1％（W／W）的水平,用建立起来的方法对进口肉骨粉,鱼粉和饲料添加剂进行了检测,结果在5批肉骨粉中检测出含有可能导致疯牛病的牛成分。     

Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real-time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S-layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex-PCR. Among them, heat treatment of samples suspended with sucrose plus non-ionic detergent was considered an effective spore disruption method because it detected up to 10(5) spores/g soil by multiplex-PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/ microliters and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real-time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between C(T) value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 10(4) spores/g soil within 3 hr by real-time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real-time PCR, avoiding time and labor-consuming preparation steps such as enrichment culturing and DNA preparation.     

Rapid DNA detection by interface PCR on nanoparticles

A novel, rapid DNA detection method based on fluorescence quenching of quantum dots (QDs) by gold nanoparticles (GNPs) through polymerase chain reaction (PCR) was developed. In proof-of-concept experiments, the length of the amplicon DNA ranging from 152 to 1003 base pairs (bp) could be determined based on quenched fluorescence intensity with 136 bp as the lower limit of effective range. And the real sample detections were also achieved successfully by this developed method. Therefore, this DNA detection method has the potential to be the powerful gene diagnostic tool.     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

OIE white spot syndrome virus PCR gives false-positive results in Cherax quadricarinatus

White spot syndrome virus (WSSV) is an intranuclear bacilliform virus (IBV) that is a serious, notifiable crustacean pathogen. The Office International des Epizooties (OIE) PCR protocol for WSSV uses primer sets initially developed by Lo et al. (1996). It yields a first-step PCR amplicon of 1441 bp and a nested PCR amplicon of 941 bp. An amplicon (941 bp) purported to specifically detect WSSV was obtained when using template DNA extracted from Cherax quadricarinatus in a WSSV PCR detection protocol recommended by the OIE. Sequencing and analysis of the 941 bp amplicon and an occasional 550 bp amplicon from C. quadricarinatus revealed no phylogenetic relationship with WSSV, and suggested a possible lack of sufficient primer specificity for WSSV in the OIE test. This suggestion was supported by the fact that the OIE outer primer sequence (146F1) was present in both the forward and reverse position of the 941 bp and the forward position of the 550 bp nested amplicons from C. quadricarinatus. As WSSV is a notifiable pathogen, the consequences of false-positive results are harsh in WSSV-free zones and can lead to incorrect quarantine and unnecessary destruction of animals. Therefore, urgent attention and revision is necessary for the current OIE PCR protocol for WSSV detection.     

The effects of temperature on detection of prey DNA in two species of carabid beetle

PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12 degrees C, 16 degrees C and 20 degrees C, and batches of beetles were subsequently frozen at time periods from 0-72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20 degrees C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.     

Persistence and mobility of a Clostridium botulinum spore population introduced to soil with spiked compost

In a recent study it could be shown that compost samples can contain Clostridium botulinum. It was investigated if C. botulinum introduced with compost into botulinum-free soil can persist and be translocated within the soil. Compost was spiked with two C. botulinum type D spore concentrations (10(3) and 10(5) spores g(-1)) and the composts were spread on an experimental site. Over a period of 939 days, samples were taken from the upper (0-5 cm) and the lower (10-30 cm) soil horizons. Physical and chemical as well as microbiological variables were measured. Clostridium botulinum spores were quantified in a culture MPN-PCR assay. On day 757 the last positive sample was obtained in the plots with the lower spore concentration (10(3) g(-1)). The bacteria were never detected in the samples taken from the lower horizons of these plots. Clostridium botulinum persisted over the whole investigation period in the plots which were treated with compost spiked with 10(5) spores g(-1). The concentrations found were between 20 and 20,000 spores g(-1) soil. The bacteria were vertically translocated and could be found in the lower soil horizons (20-2000 spores g(-1) soil) starting 70 days after the compost was spread.     

Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp.

Hepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.     

Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.     

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.     

Evaluation of amplified ribosomal DNA restriction analysis (ARDRA) and species-specific PCR for identification of Bifidobacterium species

Molecular biological methods based on genus-specific PCR, species-specific PCR, and amplified ribosomal DNA restriction analysis (ARDRA) of two PCR amplicons (523 and 914bp) using six restriction enzymes were used to differentiate among species of Bifidobacterium. The techniques were established using DNA from 16 type and reference strains of bifidobacteria of 11 species. The discrimination power of 914bp amplicon digestion was higher than that of 523bp amplicon digestion. The 914bp amplicon digestion by six restrictases provided unique patterns for nine species; B. catenulatum and B. pseudocatenulatum were not differentiated yet. The NciI digestion of the 914bp PCR product enabled to discriminate between each of B. animalis, B. lactis, and B. gallicum. The reference strain B. adolescentis CCM 3761 was reclassified as a member of the B. catenulatum/B. pseudocatenulatum group. The above-mentioned methods were applied for the identification of seven strains of Bifidobacterium spp. collected in the Culture Collection of Dairy Microorganisms (CCDM). The strains collected in CCDM were differentiated to the species level. Six strains were identified as B. lactis, one strain as B. adolescentis.     

Human MECP2 gene at Q28 arm of X chromosome as a suitable target for monitoring PCR inhibition in a nested, multiplexed HIV-1 DNA detection protocol

Human MECP2 gene located at q28 arm of X chromosome was identified as target for thermal co-amplification with HIV-1 proviral DNA of infected individuals. The selected MECP2 gene-specific primers functioned at a wide range of annealing temperature, extension time and exhibited no significant interaction with pathogen specific primers. A 466 bp PCR amplicon originating from human MECP2 gene was found to be diagnostic for inhibition-free PCR reaction when co-amplified with the HIV-1 target gene in a multiplexed, nested PCR reaction. The 5' end of the MECP2 primers were engineered to position an EcoRI restriction endonuclease site to facilitate rapid cloning in various DNA vector molecules at the corresponding EcoRI sites. Cell mass of Escherichia coli (XL1Blue) harboring the recombinant plasmid when added to pleural fluid of HIV-1 infected individuals co-infected with Mycobacterium tuberculosis, generated the diagnostic 466 bp MECP2 PCR amplicon as well as the 194 bp PCR amplicon of target gene from M. tuberculosis. The experiment underlined potential of the region spanning nucleotide position 4118099 to 4118552 of human MECP2 gene (NCBI accession number NT_011726.13) as a reliable target for multiplex PCR to accommodate a wide range of thermal cycling and multiplex reaction conditions. In both cases of this study, electrophoresis-based separation of the 466 bp MECP2 fragment and the 232 bp and 194 bp HIV-1 and M. tuberculosis fragments respectively was distinct and unambiguous. The potential of this human MECP2 gene available from human genome or recombinant plasmid as a potent target to monitor PCR inhibition for a range of different PCR reactions is discussed.     

Qualitative and quantitative analysis of genetically modified pepper

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.     

Differentiation of Listeria isolates by PCR amplicon profiling and sequence analysis of 16S-23S rRNA internal transcribed spacer loci

The 16S-23S rRNA internal transcribed spacer region (ITS) in genomic DNA from Listeria species was amplified and sequenced so as to find sequence differences that would allow rapid species and strain differentiation. Agarose gel profiles of amplicons generated with primers designed to amplify ITS loci indicated that Listeria DNA can contain at least two distinct ITS regions. The direct sequencing of the smaller of these ITS amplicons (330 bp) was found useful for the rapid and accurate differentiation of various Listeria species. On the other hand analysis of ITS amplicons generated from a total of 27 L. monocytogenes strains indicated that 4/27 of these strains could be distinguished on the basis of their ITS profile (the presence of a unique 350 bp amplicon). The lack of sequence heterogeneity in the small 333 bp amplicon did not permit rapid strain differentiation.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Enhancement of a hybridization analysis efficiency by the controlled DNA fragmentation

The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes.     

Identification of Cotton Hybrid through the Combination of PCR Based RAPD, ISSR and Microsatellite Markers

Hybrid cotton H ‘6’ and its parents G.Cot.10 (male) and G.Cot.100 (female) were studied for identification with three PCR based molecular markers, RAPD, ISSR and microsatellite. Twenty RAPD primers, nineteen ISSR primers and twenty-five JESPR cotton microsatellite loci were used. RAPD primer OPA 11 was found to be useful in differentiating parents and hybrid. Two ISSR primers, IS4 and IS7 showed polymorphism in the parents. IS4 identified a female-specific amplicon of about 500bp and IS7 identified two female-specific amplicons of about 500 and 1200bp in the hybrid H ‘6’. Microsatellite loci JESPR-2 and JESPR-17 were found to be heteroallelic for parents. JESPR-2 identified one male-specific repeat of about 850bp, while JESPR-17 detected two male-specific repeats of about 800bp and 700bp in the hybrid H ‘6’. Results indicated that using all three markers - RAPD, ISSR and SSR - in combination is faster and more reliable than using the three in isolation, for the identification of cotton hybrid.