Species-specific identification of porcine blood plasma in heat-treated chicken meatballs

This study was conducted to detect the presence of chicken and porcine DNA in meatballs using mitochondria DNA (mtDNA) of cytochrome b (cyt b) and nuclear DNA (nDNA) short interspersed nuclear element (SINE) species-specific primers, respectively. While, the mtDNA primers targeted transfer RNA-ATP8 (tRNA-ATP8) gene was used for 1 and 5% (w/w) chicken meatball spiked with commercial porcine blood plasm. Chicken meatballs spiked with 1% and 5% (v/w) fresh and commercial porcine blood plasma, respectively were prepared and heat-treated using five (n = 5) cooking methods: boiling, pan-frying, roasting, microwaving and autoclaving. Two pairs of mtDNA and nDNA primers used, produced 129 and 161 bp amplicons, respectively. Whereas, tRNA-ATP8 primers produced 212 bp of amplicon. Electrophoresis analysis showed positive results for porcine DNA at 1% and 5% (w/w or v/v) for all of the different cooking techniques, either for fresh or commercial blood plasma using SINE primers but not for tRNA-ATP8 primers. The present study has highlighted the useful of species-specific primers of SINE primers in PCR analysis for detecting porcine DNA blood plasma in heat-treated chicken meatballs.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

Consensus multiplex PCR–restriction fragment length polymorphism (RFLP) for rapid detection of plant mitochondrial DNA polymorphism

A fast, simple, and efficient approach, termed consensus multiplex PCR–RFLP, was developed and employed to detect mitochondrial (mt)DNA variation in three orchid species, Spiranthes hongkongensis, S. sinensis , and S. spiralis . Using multiplex PCR, three pairs of consensus mitchondrial primers were added simultaneously into each reaction tube to amplify three nonoverlapping introns located in the NADH dehydrogenase genes. Fragment length differences in the multiplex PCR amplicons were directly detectable between S. spiralis and the other two species. Further restriction analysis of the multiplex PCR amplicons revealed sufficient mtDNA polymorphism, suitable for phylogenetic studies at the interspecific level. This approach is well suited for large-scale population surveys of mitochondrial genome diversity in plants. Additionally, the maternal mode of inheritance of organelle genomes renders this approach valuable for rapid identification of the origin and specific parentage of hybrid or allopolyploid species.     

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100-0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.     

Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp

A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period.     

Development of a rapid,reliable and simple multiplex PCR assay for early detection of transgenic plant materials

Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR

Displacement probes have recently been described as a novel probe-based detection system for use in both quantitative real-time polymerase chain reaction (PCR) and single nucleotide polymorphism genotyping analysis. Previous reports have shown that shorter probes (23 mer) had improved detection sensitivity relative to longer probes (29 mer), with the likely reason for this effect being the improved hybridization kinetics of shorter probes. Sterically modified locked nucleic acids (LNAs) have been used to improve the design of a range of real-time PCR probes by raising the melting temperature (Tm) of the probe and enabling shorter probe designs to be considered. A displacement probe for gapdh was designed and tested successfully, and this probe was then redesigned with LNAs to an 11 mer probe. This probe showed increased detection sensitivity compared with the original 26 mer probe. To detect the widest range of displacement probe designs at maximum sensitivity, we have also developed a novel fluorescence capture two-step PCR protocol. This method produces enhanced probe quenching with a single standardized protocol ideal for high-throughput applications. The displacement probes tested produced sensitive and efficient quantitative analyses of template serial dilutions when compared with a range of commercially available predesigned real-time PCR detection systems, including TaqMan MGB probes, QuantiTect MGB probes, and LUX primers.     

Detection of Phytophthora nicotianae in Soil with Real-time Quantitative PCR

Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora , the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/μl in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/μl and in TaqMan PCR 1.2 fg/μl, and real-time PCR was 10⁴–10⁵ times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/μl. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method.     

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at –20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (146 bp) and a nuclear DNA(nDNA) locus (200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26–88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.     

A decreased mitochondrial DNA content is related to insulin resistance in adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross-sectional, population-based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA-IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real-time quantitative PCR method. Genotyping for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) (pro12Ala), PPAR- gamma coactivator-1alpha (PGC-1alpha) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR-based restriction fragment length polymorphism. Long-extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P<0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: -0.18, P<0.02), glucose (R: -0.21, P<0.008), and uric acid (R: -0.18, P<0.03). Genotypes for the PPAR- gamma, PGC-1alpha, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long-extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.     

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.     

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.     

Quantification of Pyrenophora teres in infected barley leaves using real-time PCR

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

A species-specific real-time PCR assay for identification of three lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.     

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。