Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.     

Simple procedure for rapid identification of Vibrio cholerae from the aquatic environment

Biochemical tests commonly used to screen for Vibrio cholerae in environmental samples were evaluated, and we found that a combination of alkaline peptone enrichment followed by streaking on thiosulfate citrate bile salts sucrose agar and testing for arginine dihydrolase activity and esculin hydrolysis was an effective rapid technique to screen for aquatic environmental V. cholerae. This technique provided 100% sensitivity and > or =70% specificity.     

A gyrB-targeted PCR for rapid identification of Paenibacillus mucilaginosus

Paenibacillus mucilaginosus, one of the typical silicate bacteria, has long been used as a biofertilizer in agriculture and has recently shown potential in bioleaching and wastewater engineering. There has been considerable research involving the isolation of P. mucilaginosus for various utilizations; therefore, rapid identification of this species is of great interest. Herein, we describe a specific polymerase chain reaction (PCR) method developed for a rapid identification of P. mucilaginosus, which might provide potential utilization in the investigation of populations, detection of biofertilizers, and identification of novel isolates on a large scale. A gyrB-targeted species-specific primer pair, F2 (5′-ACG GAT ATC TCC CAG ACG TTC AT-3′) and R5 (5′-ACG GGC ACG CTG CGC CTG TAC G-3′), was successfully designed to selectively amplify a 519-bp amplicon from P. mucilaginosus. Good specificity was demonstrated by both reference strains and total soil deoxyribonucleic acid, from which only the gyrB gene of P. mucilaginosus was amplified. The detection limit was 4–10 cells per assay. Using the culture-PCR method, 20 of 26 soil isolates on a nitrogen-free medium were rapidly identified as P. mucilaginosus, which was confirmed by sequencing of the gyrB gene.     

Development of a PCR for identification of Naegleria fowleri from the environment.

A species-specific PCR for the identification of Naegleria fowleri was developed. In sensitivity studies, 10 trophozoites or cysts and 1 trophozoite or cyst could be detected after 35 and 45 cycles, respectively. In conjunction with a rapid DNA isolation method, this PCR was used to identify N. fowleri directly from primary cultures of environmental samples.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.     

PCR and blot hybridization for rapid identification of Haloferax species

Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed. Two variable regions of the 16S rDNA in Haloferax spp. were selected as genus-specific primers for the PCR assay and hybridization probe. Five genera of halophilic Archaea and Escherichia coli were examined as outside groups. Using this approach, all strains of Haloferax spp. were positive. In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative. In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach. This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp. in hypersaline environments.     

Multi-primer target PCR for rapid identification of bovine DRB3 alleles

Multi-primer target polymerase chain reaction (MPT-PCR) is a rapid method for the identification of specific BoLA-DRB3 alleles. In a single PCR reaction, the presence of two alleles associated with increased risk, DRB3.2*23 (DRB3*2701-2703, 2705-2707) and decreased risk, DRB3.2*16 (DRB3*1501, 1502), of mastitis in Canadian Holstein can be detected. Two outer primers amplify exon 2 of DRB3. Simultaneously, two inner, allele-specific primers amplify individual alleles. Initially, 40 cows previously typed by PCR-restriction fragment length polymorphism (PCR-RFLP) were genotyped using the multi-primer approach. An additional 30 cows were first genotyped by multi-primer target PCR, then by PCR-RFLP. All animals were correctly identified and there were no false positives. This technique can readily be modified to identify other BoLA alleles of interest.     

设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌

乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。     

Fluorescence-assisted image analysis of freshwater microalgae

We exploit a property of microalgae-that of their ability to autofluoresce when exposed to epifluorescence illumination-to tackle the problem of detecting and analysing microalgae in sediment samples containing complex scenes. We have added fluorescence excitation to the hardware portion of our microalgae image processing system. We quantitatively measured 120 characteristics of each object detected through fluorescence excitation, and used an optimized subset of these characteristics for later automated analysis and species classification. All specimens used for training and testing our system came from natural populations found in Lake Biwa, Japan. Without the use of fluorescence excitation, automated analysis of images containing algae specimens in sediment is near impossible. We also used fluorescence imaging to target microalgae in water samples containing large numbers of obtrusive nontargeted objects, which would otherwise slow processing speed and decrease species analysis and classification accuracy. Object drift problems associated with the necessity to use both a fluorescence and greyscale image of each microscope scene were solved using techniques such as template matching and a novel form of automated seeded region growing (SRG). Our system proved to be not only user-friendly, but also highly accurate in classifying two major genera of microalgae found in Lake Biwa-the cyanobacteria Anabaena spp. and Microcystis spp. Classification accuracy was measured to be over 97%.     

Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans

Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular Dde I restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans . Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the Dde I restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans . We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa , weilii , and inadai , but also simplified a previous PCR-RFLP protocol.     

Immuno capture PCR for rapid and sensitive identification of pathogenic Bacillus anthracis

Immuno capture PCR (IPCR) is a technique capable of detecting the pathogens with high specificity and sensitivity. Rapid and accurate detection of Bacillus anthracis was achieved using anti-EA1 antibodies to capture the cells and two primer sets targeting the virulence factors of the pathogen i.e., protective antigen (pag) and capsule (cap) in an IPCR format. Monoclonal antibodies specific to B. anthracis were generated against extractable antigen 1 protein and used as capture antibody onto 96 well polystyrene plates. Following the binding of the pathogen, the DNA extraction was carried out in the well itself and further processed for PCR assay. We compared IPCR described here with conventional duplex PCR using the same primers and sandwich ELISA using the monoclonal antibodies developed in the present study. IPCR was capable of detecting as few as 10 and 100 cfu ml^?1 of bacterial cells and spores, respectively. IPCR was found to be 2–3 logs more sensitive than conventional duplex PCR and the sandwich ELISA. The effect of other bacteria and any organic materials on IPCR was also analyzed and found that this method was robust with little change in the sensitivity in the presence of interfering agents. Moreover, we could demonstrate a simple process of microwave treatment for spore disruption which otherwise are resistant to chemical treatments. Also, the IPCR could clearly distinguish the pathogenic and nonpathogenic strains of B. anthracis in the same assay. This can help in saving resources on unnecessary decontamination procedures during false alarms.     

Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

Rapid harvesting of freshwater microalgae using chitosan

In this study, chitosan was used as a flocculant to harvest freshwater microalgae Chlorella vulgaris. The recovery efficiency of C. vulgaris was tested at various chitosan concentrations. 120 mg/L of chitosan showed the highest efficiency (92 ± 0.4%) within 3 min. The maximum concentration factor of 10 was also achieved at this dose of chitosan. The harvesting efficiency was pH dependent. pH 6.0 showed the highest harvesting efficiency (99 ± 0.5%). Measurement of zeta-potential confirmed that the flocculation was induced by charge neutralization. This study showed that a biopolymer, chitosan, can be a promising flocculant due to its high efficacy, low dose requirements, and short settling time.     

A rapid DNA extraction method for PCR identification of fungal indoor air contaminants

Following air sampling fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and Polymerase Chain Reaction (PCR) applications. The methodology described is both rapid and cost effective for use with multiple fungal organisms.     

Real time PCR hybridization for the rapid and specific identification of Francisella tularensis

Tularemia is highly infectious and fatal zoonotic disease caused by Gram negative bacteria Francisella tularensis. The necessity to undergo medical treatment in early phase of illness in humans and possibility of making use of bacterial aerosol by terrorists in an attack create an urgent need to implement a rapid and effective method which enables to identify the agent. In our study two primers FopA F/R and hybridization probes FopA S1/S2 designed from fopA gene sequence, were tested for their potential applicability to identify F. tularensis. In this research 50 strains of F. tularensis were used and the test gave positive results. Reaction specificity was confirmed by using of non-Francisella tularensis bacterial species. The results obtained in the real-time PCR reaction with primers Tul4 F/R and hybridization probes Tul4 S1/S2, designed from tul4 gene, were comparable to the results from previous experiment with fopA - primers set. Investigation of fopA and tul4 primers and hybridization probes properties revealed characteristic Tm (melting temperature) value of the products--61 degrees C and 60 degrees C, respectively. Detection sensitivity was remarkably higher when fopA primers set was used 1 fg/microl, and for tul4 primers set, minimal detectable concentration is 10 fg/microl.     

Metabolites from freshwater aquatic microalgae and fungi as potential natural pesticides

Microorganisms are recognized worldwide as the major source of secondary metabolites with mega diverse structures and promissory biological activities. However, as yet many of them remain little or under-explored like the microbiota from freshwater aquatic ecosystems. In the present review, we undertook a recompilation of metabolites reported with pesticidal properties from microalgae (cyanobacteria and green algae) and fungi, specifically from freshwater aquatic habitats.