Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.     

Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

Parentage analyses in ant colonies using simple sequence repeat loci

In ants of the genus Myrmica , female progeny may be the offspring of one to several different queens. In addition, both workers and queens are capable of producing haploid male offspring. Even in such complex colonies, parentage can be assigned on the basis of genotypic variation at highly polymorphic simple sequence repeat loci. Methods are described for isolating and screening dinucleotide repeat loci in ants. Three independent loci, Myrt2, Myrt3 and Myrt4, show expected heterozygosities of 0.94, 0.92 and 0.95, respectively. When used in parallel these loci should be sufficient to establish parentage in the vast majority of screened colonies. An initial screening indicates that males are produced by workers in the subalpine ant Myrmica 'near tahoensis'.     

Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction

Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5-GTTTCGCTCC-3) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg²⁺, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints.The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms.     

Rapid identification of Triticeae genotypes from single seeds using the polymerase chain reaction

An easy and quick protocol has been developed for DNA analysis via PCR. Single cereal endosperm or small leaf pieces can be separately processed in several PCR reactions. The resultant PCR patterns are equivalents to those obtained with standard DNA extraction protocols using either specific or random primers. Intra-and inter-specific variability can be detected. This method allows the analysis of a large number of individuals in early stages prior to the plant sowing.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

Diagnosis of bovine freemartinism by the polymerase chain reaction method

Summary. Using sex chromosome specific primers, leucocyte chimaerism in heterosexual bovine female twins was identified by combining polymerase chain reaction (PCR) and restriction enzyme digests.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Miniaturization of polymerase chain reaction

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.     

High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants

Single pollen grain polymerase chain reaction （PCR） has succeeded in several species, however only limited numbers of pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput （HT） procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR is reported. The HT procedure involved the collection of individual pollen grains by a pair of special forceps and the lysis of pollen grains in a heated alkali/detergent solution followed by neutralization with a tris-ethylenediamine tetraacetic acid （TE） buffer. These resulting template solutions yielded PCR reactions involving the 5S ribosomal RNA intergenic spacers, randomly amplified polymorphic DNA, and simple sequence repeats markers. Using this procedure, one person with experience could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also significantly benefit studies in species that are difficult subjects for classical genetic research.     

Absence of restriction site variation in the mitochondrial ND5 and ND6 genes of Atlantic salmon amplified by the polymerase chain reaction

There was no restriction site variation in Atlantic salmon mitochondrial DNA in an amplified DNA segment beginning in the ND5 gene and terminating in the ND6 gene, although seven haplotypes were observed when the complete molecule was restricted.     

Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction

Abstract We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmania species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.     

数字聚合酶链反应及其在感染性疾病中的应用

数字聚合酶链反应(polymerase chain reaction,PCR)采用与定量PCR相同的荧光化学原理和不同的数学原理来实现对靶标核酸序列的绝对定量,其摒弃了对外部参照的依赖,同时具有更高的数据精密度,提高了重复性和再现性。数字PCR的应用涵盖生命科学众多领域,特别是在医学检验领域,其对疾病相关核酸分子标记的准确分析,为疾病的早期诊断、进展监测、疗效评估提供了动态量化指标。数字PCR的出现将推动基于核酸扩增技术的分子生物学检测迈入精准定量阶段。本文就数字PCR尤其是微滴式数字PCR在感染性疾病中的应用进展及前沿进行综述。     

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.     

Characterization of six microsatellite primers for the grey fox (Urocyon cinereoargenteus)

We describe polymerase chain reaction (PCR) primers and conditions to amplify one dinucleotide and five tetranucleotide microsatellite DNA loci isolated from the grey fox (Urocyon cinereoargenteus). The PCR primers were tested on nine to 12 individuals collected from the Department of Energy's Savannah River site (Aiken, SC, USA). The grey fox microsatellite primers developed had three to 10 alleles per locus that yielded observed heterozygosities ranging from 0.222 to 0.889.     

Sixteen new simple sequence repeat markers from Brassica juncea expressed sequences and their cross‐species amplification

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 16 expressed sequence tags (EST)‐SSR markers from Brassica juncea and their cross‐amplification across Brassica species. Sixteen primer pairs were assessed for polymorphism in all genomes of the diploid and amphidiploid Brassica species. The markers show reliable amplification, considerable polymorphism and high transferability across species, demonstrating the utility of EST‐SSRs for genetic analysis of brassicas.