Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications.

We have developed a novel double Amplification Refractory Mutation System (double ARMS) using a highly polymorphic region 5' to the human delta-globin gene as a model system. The double ARMS approach involves using two allele-specific ARMS primers simultaneously during DNA amplification by the polymerase chain reaction (PCR). The resulting system is highly sensitive and more specific than single ARMS. In addition, this approach enables the elucidation of the relationship of polymorphic sites on the same chromosome and thus allows the direct determination of haplotypes. We have also demonstrated that this system can be used in conjunction with inverse PCR, the resulting double ARMS inverse PCR (DARMSI-PCR) may allow haplotype determination on polymorphic sites which are separated further apart than the length limit imposed by PCR. The double ARMS approach has numerous other applications in molecular biology including HLA typing, virology, forensic pathology and the investigation of the phenomenon of chimerism following bone marrow transplantation.     

Usefulness of strb1 and 16S rDNA-targeted PCR for detection of Streptomyces spp. in environmental samples

In this study, we revealed rapid detection of streptomycin-producing Streptomyces spp. by extraction of total soil DNA from 14 soil samples using a modified lysis method followed by PCR amplification ofa genus-specific sequence in the Streptomyces' 16S rDNA gene. DNA band of the expected size (438 bp) was seen with all the samples. Additionally, specific amplification of the streptomycin-coding gene (strb1) directly from soil revealed the presence of a single DNA band of 940 bp. These results indicate that PCR-amplification of Streptomyces specific genes could be used for direct detection of streptomycin-producing Streptomyces species from soil.     

四引物扩增受阻突变体系PCR快速测定绵羊 BMPR-IB基因型方法的建立

四引物ARMS PCR是检测SNP有效、快速、简便的方法.绵羊BMPR-lB基因是控制Booroola绵羊多胎性状的主效基因,此研究目的在于建立一种对BMPR-IB基因四引物ARMS PCR检测方法.根据四引物ARMS PCR技术原理,在绵羊BMPR-IB基因突变位点(A746G)设计一对特异性引物,并在突变点两侧设计一对参照引物,用来扩增含有突变点的DNA片段,可在一步PCR反应中根据电泳图谱准确判断绵羊个体的BMPR-IB基因型,对比PCR-RFLP检测结果表明,所建立的方法简单,操作简便,大大提高了检测效率.     

Detection of His1069Gln mutation in Wilson disease by bidirectional PCR amplification of specific alleles (BI-PASA) test

Wilson disease (WD) is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase, ATP7B. The majority of known mutations affecting this gene are frequent in different populations, which may help to introduce rapid diagnostic procedures based on direct DNA analysis into routine clinical practise. The His1069Gln mutation in exon 14 is the most frequent one, accounting for 30-60% of all mutations in Caucasian patients. The aim of the present work was to introduce DNA-based direct analysis into routine molecular screening for the above mutation in Slovak WD patients and to assess its frequency in patients as well as in a control population. Twenty seven clinicaly diagnosed patients from twenty five families, twenty relatives of index patients and three hundred and six control DNA samples were tested using two different DNA-based methods: the earlier described amplification created restriction site (ACRS) for Alw21I in combination with nested PCR and the amplification refractory mutation system (ARMS). In 18 of 25 unrelated patients (72%), the mentioned genetic defect was present in at least one copy. In ten of them (40%), the above mutation was detected in homozygous and in eight individuals (32%) in heterozygous state. In seven WD patients (28%), this mutation was not detected. The allele frequency of His1069Gln in Slovak patients with WD was 56%, which was higher as reported in other populations. In a control group of 306 random DNA samples (612 alleles), the His1069Gln mutation was observed in 3 samples (carrier frequency 1%; allele frequency 0.49%). These frequencies correspond to figures observed in different population of European origin. Taken together, we have provided further evidence that the His1069Gln mutation is the prevalent ATP7B mutation in central-european WD patients. Although both methods used in this study worked in our hands reliably, there are in every-day use some drawbacks and limitations inherent to them (PCR reactions in two tubes, possibility of star activity or not complet digestion by restriction endonuclease, etc.). Therefore we developed a simpler, cost effective and rapid DNA diagnostic test based on bidirectional amplification of specific alleles (BI-PASA), which enables detection of homozygotes (wild and mutant) and heterozygotes, respectivelly, in one PCR reaction. The test was highly sensitive and specific, yielding no false-positive or false-negative results. Its reliability and discriminating power was tested on samples of 27 WD patients and 120 random control DNA's, previously genotyped by above mentioned methods. Comparing results of BI-PASA with ACRS and ARMS tests showed 100% concordance.     

Guidelines for the Tetra-Primer ARMS–PCR Technique Development

The tetra-primer amplification refractory mutation system–polymerase chain (ARMS–PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS–PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl₂. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS–PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.     

Investigation of sensitivity,specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR,based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

Purpose

Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients.

Methods

Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing.

Results

All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results.

Conclusion

Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.     

Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis

Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10–12% of wild-type DNA.     

A novel SERRS sandwich-hybridization assay to detect specific DNA target

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.     

四引物扩增受阻突变体系PCR技术及其在动植物遗传育种研究中的应用

四引物扩增受阻突变体系PCR(Tetra-primer amplification refractory mutation system PCR,Tetra-primer ARMS PCR)技术是一种在普通PCR基础上发展起来的单核苷酸多态性(SNP)分型技术。该项技术综合了扩增受阻突变体系(Amplification refractory mutation system,ARMS)和四引物PCR(tetra-primer PCR)技术的优点,是对等位基因特异性PCR法的改良。它具有操作简便、分型快速、费用低廉等特点,在国内外生命科学领域尤其是遗传育种领域的应用越来越广泛。本文介绍了四引物扩增受阻突变体系PCR的技术原理及优势、结果检测手段和反应体系改进方法,并在此基础上对该技术在遗传育种研究中的应用进行综述。     

Use of molecular biology methods for diagnosing fungal infections

A total of 130 various clinical materials taken from 48 children with suspected systemic fungal infection were used for the study. Clinical samples were tested by use of classical mycological procedures well as by use of molecular technique (PCR assay). The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification. Fifty seven samples (48%) were positive for Candida albicans and eighty four (68%) by use of PCR. It should be stressed that 4 blood samples, 21 urine samples and 5 other samples were positive by use of molecular technique, only. PCR is sensitive and rapid method for detection and identification of Candida albicans from clinical materials of children with fungal infection. This technique can be applied for monitoring presence of fungal DNA in tested samples during antifungal therapy.     

Comparison of three methods for DNA extraction from bone remains

Extraction of amplifiable DNA is a frequent problem when working with degraded specimens like bone samples. The possibility of obtaining as much information as possible from these samples has a particular significance in many forensic investigations. The present investigation was aimed to assess the efficiency of three organic extraction methods for purifying amplifiable DNA from bone samples. The amount of nucleic acids obtained, the success rate in the amplification of DNA microsatellite (STR) markers and amelogenin by PCR, the influence of PCR inhibitors and environmental conditions, and where the samples were found before their processing in the laboratory, were all evaluated in this investigation for the three methods. Results showed that method A (a modification of FBI method for DNA extraction) performed better in producing not a higher amount but a better quality amplifiable DNA, in comparison with the other two methods evaluated. It was also demonstrated that the quality of the DNA to be amplified by PCR was influenced by the presence of inhibitors and/or contaminants and the environmental conditions where the bone sample was taken from. The worst conditions were observed from aquatic environments. The results suggest that the implementation of some specific modifications in the method A (use of purification columns, reliable quantification methods and different dilutions) would help to obtain better DNA extracts intended to be used in different molecular identification tests.     

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

Rapid purification of DNA from soil for molecular biodiversity analysis

A rapid DNA extraction method utilizing a bead-beating machine is described. High molecular weight DNA could be extracted from up to 24 samples in less than 2 h. The DNA was suitable for direct use in the polymerase chain reaction (PCR). Both prokaryotic and eukaryotic cells were successfully lysed by the method, established using primers specific for these groups. The small subunits ribosomal RNA (rRNA) spacer region from both eukaryotes and eubacteria could be readily amplified with DNA from different soils generating different amplification patterns.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.     

Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.     

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.     

Cloning and sequencing of the histidine decarboxylase genes of gram-negative,histamine-producing bacteria and their application in detection and identification of these organisms in fish

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.     

Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.