Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles.

A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryI, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes.     

A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR) and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.     

Plasmodium falciparum: a simple polymerase chain reaction method for differentiating strains.

Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difficulties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 microliters of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA-2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory.     

Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer.

A method is described for preparing site-specific mutants using a polymerase chain reaction (PCR) based protocol. The protocol requires a single mutant primer, and has been used to introduce mutations into DNA fragments ranging in size from 200 bp to 1569 bp in length in the GM-CSF, beta-actin, human growth hormone and erythropoietin genes. Sequence analysis of PCR derived mutant fragments shows an error rate of less than one bp change per 1500 bp incorporated. Single base pair mutations have been introduced into these genes which create unique restriction sites. We demonstrate that these mutant templates may be used for competitive PCR to quantitate mRNA and DNA. This method thus offers a rapid means for producing competitive templates for use in quantitative PCR.     

Use of polymerase chain reaction to identify a leucyl tRNA in Streptomyces coelicolor

Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor. Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA^Leu_CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S. coelicolor, the bldA encoded tRNA^Leu_UUA. Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation [Leskiw et al., 1993. J. Bacteriol., 175, 1995–2005].     

Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction.

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.     

Full-length cDNA cloning utilizing the polymerase chain reaction, a degenerate oligonucleotide sequence and a universal mRNA primer.

A full-length cDNA was selectively amplified by the polymerase chain reaction (PCR) utilizing a primer pair consisting of a "universal" 21-base synthetic deoxyoligonucleotide (oligo dT 17GGCC) and a specific degenerate deoxyoligonucleotide sequence (DOS) derived from the N-terminal amino acid sequence. This double-stranded amplified cDNA was uni-directionally cloned into M13mp19 utilizing two restriction sites that had been previously incorporated into the termini of the universal and specific DOS primers. Cloning of the specific cDNA via this PCR amplification with the universal/specific DOS primer pair approach was confirmed by screening with a second DOS contiguous with the DOS employed to prime second (sense)-strand cDNA synthesis. This technique allows for the selective full-length cDNA cloning of low-abundance mRNAs from a single-protein sequence determination.     

A polymerase chain reaction screen of field populations of Heliothis virescens for a retrotransposon insertion conferring resistance to Bacillus thuringiensis toxin

The evolution of pest resistance to transgenic crop plants producing insecticidal toxins from Bacillus thuringiensis (Bt) Berliner poses a continuing threat to their sustainable use in agriculture. One component of the U.S.-wide resistance management plan for Bt cotton, Gossypium hirsutum L., involves monitoring the frequency of resistance alleles in field populations. However, existing methods are expensive and may not detect recessive resistance alleles until their frequencies are too high for countermeasures to be effective; therefore, more sensitive methods are needed. The first Bt resistance-causing mutation described at the molecular level was a retrotransposon insertion into the gene encoding a 12-cadherin-domain protein expressed in the midgut of larval Heliothis virescens (F.). We report the first large-scale screen for this mutation using a polymerase chain reaction (PCR)-based approach on >7,000 field-collected individuals. The specific insertion was not detected in any of these samples, nor was it detected in three progeny-tested, field-caught males thought to carry a Bt resistance gene. Unlike the targets of many chemical insecticides where a limited number of resistance-causing mutations compatible with viability can occur; a very large number of such mutations seem possible for the 12-cadherin-domain gene. However, even if these mutations are viable in the laboratory, they may not threaten the effectiveness of transgenic crops because of a high fitness cost in the field. The challenge remains to detect the subset of possible resistance-conferring alleles that are still rare but are viable in the field and increasing due to selection by Bt cotton. This situation will complicate PCR-based Bt resistance monitoring strategies.     

The use of the polymerase chain reaction to identify porcine isolates of Trichinella.

A method was developed to identify domestic isolates of Trichinella using the polymerase chain reaction. Oligonucleotide primers, based on the repetitive DNA sequence (pPRA) from the P1 isolate of Trichinella, were used to amplify genomic DNA from 13 domestic isolates and tested against sylvatic isolates of Trichinella. Pattern differences were observed among domestic isolates, indicating divergence of this repetitive sequence. The primers were specific for domestic Trichinella as no amplification was detected for sylvatic isolates or Trichinella pseudospiralis. It was possible to identify an isolate from a single larva following digestion or in situ in muscle tissue.     

Combination primer polymerase chain reaction for multi-site mutagenesis of close proximity sites.

We describe a rapid and efficient polymerase chain reaction procedure for multi-site-directed mutagenesis for cases in which the sites to be mutated are in close proximity. The combination primer polymer chain reaction method is based on a multi-site directed mutagenesis protocol together with a splicing by overlapping extension polymerase chain reaction protocol. several different combinations of multiple mutations were successfully performed with this method and are reported in this study.     

DNA sequencing by a subcloning-walking strategy using a specific and semi-random primer in the polymerase chain reaction.

In its basic concept, in vitro DNA amplification by the polymerase chain reaction (PCR) is restricted to those instances in which segments of known sequence flank the fragment to be amplified. Recently, techniques have been developed for amplification of unknown DNA sequences. These techniques, however, are dependent on the presence of suitable restriction endonuclease sites. Here, we describe a strategy for PCR amplification of DNA that lies outside the boundaries of known sequence. It is based on the use of one specific primer, homologous to the known sequence, and one semi-random primer. Restriction sites in the 5' proximal regions of both primers allow for cloning of the amplified DNA in a suitable sequencing vector or any other vector. It was shown by sequence analysis that the cloned DNA fragments represent contiguous DNA fragments that are flanked at one side by the sequence of the specific primer. When omitting the semi-random primer, a single clone was obtained, which originated from PCR amplification of target DNA by the specific primer in both directions.     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.     

Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.     

Differentiation of capripoxvirus species and strains by polymerase chain reaction

A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine stains used in Russia and countries of the former Soviet Union from epizootic isolates of sheep pox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.     

Differentiation of capripoxvirus species and strains by polymerase chain reaction

A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine strains used in Russia and countries of the former Soviet Union from epizootic isolates of sheeppox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.     

Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.