DNA Extraction Procedure Research Articles

The presence of Diptera larvae in human bones

The presence of Diptera larvae in the bone marrow cavities so far has been the subject of very few scientific papers. The Japanese authors found the 3rd instar larvae of the family Piophilidae (Diptera) in human skeletal remains (marrow space of the right femur) during a forensic autopsy. In turn, German authors discovered larvae of Stearibia nigriceps and Liopiophila varipes (Diptera, Piophilidae) within the medullary cavity of deer bones (tibia, metacarpus, humerus) during a forensic entomological and anthropological field study. The aim of this study is to present our own experiences with the presence of larvae in human bones sent to our Department, mainly by the prosecutors, during criminal investigations. The results of morphological examination of the bones and entomological analysis of discovered larvae were presented. We found numerous larvae of Stearibia nigriceps and surprisingly single third instar larva of Lucilia sericata and puparium of Sarcophaga argyrostoma. Our findings suggest that Diptera larvae are able to penetrate the bones of human corpse. Forensic geneticists may find such valuable entomological evidence (hidden larvae population) during routine preparation of the bones before DNA extraction procedure. In this case, the larvae should be always preserved for further possible forensic entomological studies.

On-site detection of Xylella fastidiosa in host plants and in "spy insects" using the real-time loop-mediated isothermal amplification method.

A recent severe outbreak of Xylella fastidiosa associated with ‘olive quick decline syndrome’ (OQDS) was reported in Apulia (Southern Italy). In this study an on-site real-time loop-mediated isothermal amplification (real-time LAMP) was developed for detecting X. fastidiosa in host plants and insects. A marked simplification of the DNA extraction procedure was obtained by heating the samples in a portable Smart-Dart device and using an optimized enhancer reaction buffer. The connection to a tablet or Smartphone allowed to visualize the results of the reaction in real time. Compared to PCR and ELISA, with which it showed comparable results in terms of sensitivity and reliability in the X. fastidiosa detection, this simplified real-time LAMP procedure proved to be “user friendly”, displaying the advantages to be an on-site detection method of easy handling, rapid execution and low cost.

Crime scene DNA sampling by wet-vacuum applying M-Vac

A possible alternative to conventional stain recovery by swabbing, taping or cutting, is the M-Vac wet-vacuum instrument (M-Vac Systems Inc.). We have evaluated M-Vac for sampling of dried saliva on porous and non-porous surfaces, shed cells on clothes and touch DNA. M-Vac gave significantly higher DNA yields for dried saliva stains on laminated wood, compared with cotton swabs (average DNA concentrations 1.14 vs. 0.57ng/μL, p=0.02). For stains on glass, M-Vac and cotton swabs gave comparable DNA yields. Additionally, M-Vac retrieved three times as much DNA from saliva stains on cotton fabric (T-shirt) compared with saliva on towels (terry cloth), showing that the absorption properties of the surface affect wet-vacuum sampling. M-Vac was also applied for retrieving wearer DNA from clothes, enabling generation of complete DNA profiles from denim jeans, leggings and cotton T-shirt. A mixed DNA profile was retrieved from an “aggressor” pressing a hand against the shoulder area of a worn T-shirt. Since the major component of the obtained mixed DNA profile was from the wearer, M-Vac may not be ideal for touch DNA sampling of clothes. Wet-vacuum sampling requires a fairly large instrument, trained users and DNA extraction procedures handling large sample volumes. The complexity of M-Vac sampling prevents it from being extensively used, but in specific and important cases it can be a valuable sampling tool.

Comparison of commercial DNA kits and traditional DNA extraction procedure in PCR detection of pork in dry/fermented sausages

In the present study four commercially available DNA extraction kits (Wizard® Genomic DNA Purification Kit, High Pure PCR Template Kit, DNeasy mericon Food and GeneJET PCR Purification Kit), as well as standard phenol/chloroform isolation technique have been evaluated regarding their concentration, purity and suitability for amplification of porcine DNA in dry/fermented sausages. The isolates were assessed for quantity and quality using spectrophotometer (IMPLEN GmbH, Germany). To verify template usability and quality of isolated DNA, the polymerase chain reaction (PCR) targeting at porcine cytochrome b by species specific primers was used. The comparison of extraction methods revealed satisfactory efficiency and purity of all extraction kits, while with standard phenol/chloroform isolation method high concentrations of DNA with low A260/280 were obtained. However, all the investigated techniques proved to be suitable for identification of porcine DNA in dry/fermented sausage. Thus, the standard phenol/chloroform DNA extraction method, as the cost- effective one, can be recommended as a good alternative to more expensive isolation kits when investigating the presence of pork DNA in dry/fermented meat products.

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis.

BackgroundUrogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis.Methodology/Principal FindingsA LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%).Conclusions/SignificanceWe have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.

16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice.

BackgroundCharacterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning.ResultsThe bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1–V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised “universal” PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers.ConclusionsThis work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0087-4) contains supplementary material, which is available to authorized users.

The detection of Atlantic cod (Gadus morhua) using loop mediated isothermal amplification in conjunction with a simplified DNA extraction process

Atlantic cod (Gadus morhua) is a commercially important species of white fish, and one of three species legally identifiable as cod in the UK. Mislabelling of G. morhua does occur, as does the substitution of G. morhua for less expensive species. Sensitive molecular tests based on PCR have been developed for this species, but they have limitations, including the need for expensive thermal cycling equipment, and complex DNA extraction procedures. A loop mediated isothermal amplification (LAMP) assay was designed for the G. morhua cytochrome b gene, which was capable of detecting 0.1% w/w G. morhua in a homogenised raw fish mix. The LAMP assay was also able to detect G. morhua DNA when a rapid sample preparation was used, involving heating 100 mg of fish in a 1 ml aliquot of water and testing the supernatant, showing a higher tolerance of amplification inhibitors than a PCR assay. The LAMP assay did not generate a positive result when challenged with a range of non-target species, including Gadus macrocephalus, and Gadus chalcogrammus, indicating a high level of specificity. Direct detection of a positive reaction using propidium iodide was also demonstrated.

Comparison of manual and semi-automatic DNA extraction protocols for the barcoding characterization of hematophagous louse flies (Diptera: Hippoboscidae)

The barcoding of life initiative provides a universal molecular tool to distinguish animal species based on the amplification and sequencing of a fragment of the subunit 1 of the cytochrome oxidase (COI) gene. Obtaining good quality DNA for barcoding purposes is a limiting factor, especially in studies conducted on small-sized samples or those requiring the maintenance of the organism as a voucher. In this study, we compared the number of positive amplifications and the quality of the sequences obtained using DNA extraction methods that also differ in their economic costs and time requirements and we applied them for the genetic characterization of louse flies. Four DNA extraction methods were studied: chloroform/isoamyl alcohol, HotShot procedure, Qiagen DNeasy(®) Tissue and Blood Kit and DNA Kit Maxwell(®) 16LEV. All the louse flies were morphologically identified as Ornithophila gestroi and a single COI-based haplotype was identified. The number of positive amplifications did not differ significantly among DNA extraction procedures. However, the quality of the sequences was significantly lower for the case of the chloroform/isoamyl alcohol procedure with respect to the rest of methods tested here. These results may be useful for the genetic characterization of louse flies, leaving most of the remaining insect as a voucher.

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

AimTo present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina.MethodsSamples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach.ResultsA total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications.ConclusionDNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them.

A comparison of DNA extraction methods for biodiversity studies of eukaryotes in marine sediments

There is increasing interest in understanding the diversity of eukaryotic microorgan- isms living in marine sediments, particularly to assess the effects of anthropogenic activities. Sequencing technologies generate high-resolution data for biodiversity studies that are useful for environmental monitoring. However, there are challenges in coupling classical monitoring with new sequencing technology and, consequently, there is a requirement for stringent optimization and standardization of any new protocol. Sample preparation is a critical factor because errors introduced during this step will severely affect further analyses and conclusions. This is particu- larly important in studies where biodiversity between different samples is compared, such as in environmental monitoring programs. Several protocols for extracting genomic DNA from soil and sediment samples have been developed, but most are optimized for prokaryotes in terrestrial soils and may therefore not be optimally adapted for investigating benthic marine eukaryotes. In this study, we compared existing and modified genomic DNA extraction methods on 2 different mar- ine sediments with the aim to find an optimal protocol for processing a high number of sediment samples for further sequencing analysis. The protocols were evaluated based on quantity and quality of genomic DNA and recovered biodiversity. Results indicated significant variations in overall genomic DNA yield and purity among protocols. Further, our data suggest an effect of genomic DNA extraction procedure on eukaryotic diversity profiles, particularly for sediments with a high content of silt and clay.

Grapevine yellows: Comparison of different procedures for DNA extraction and amplification with PCR for routine diagnosis of phytoplasmas in grapevine

In order to devise a procedure to be used as reference for detection of grapevine phytoplasmas and monitoring of Flavescence doree, 12 combinations comprising three methods of plant DNA extraction and 4 procedures for amplification in polymerase chain reaction of phytoplasma DNA were examined in parallel using the same plant tissues infected with phytoplasmas. In a first series tissues of periwinkles ( Catharanthus roseus ) infected with phytoplasma isolates of the Elm yellows group (16SrV) and maintained in the greenhouse, were used. In a second series tissues of grapevines ( Vitis vinifera ) naturally infected with Flavescence doree or Palatinate grapevine yellows phytoplasma were used. The DNA preparations obtained with each of the three extraction procedures were used undiluted or serially diluted, as target DNA in the 4 nested-polymerase chain reactions. The results showed differences in the efficiency among different methods of extraction as well as in the sensitivity among the DNA amplification procedures, which improved when DNA extracted from field grapevines was diluted. After additional comparative validation on numerous field-collected samples of GY-affected grapevines, the quickest extraction procedure was selected for use in routine diagnosis, with nested-PCR amplification either of ribosomal DNA or of the FD9 DNA fragment specific for Flavescence doree and other 16SrV group phytoplasmas.

Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol–chloroform–isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.

Identification of herbal components in decoctions and medicinal granules by DNA markers

Genomic DNA provides a rich source of material for species identification. However, molecular authentication is usually regarded as unsuitable for herbal decoctions and medicinal granules, as DNA is highly degraded. We set forth to investigate the availability of DNA in these products using PCR and DNA sequencing. Decoctions with Panax ginseng (Ginseng) or P. quinquefolius (American ginseng), two classical ginseng prescriptions with multiple herbal materials, and a commercial ginseng soup were tested. The DNA extraction procedure, sample pulverization, and boiling time were examined. Results showed: (1) Sample pulverization before boiling improved PCR detection. (2) Prolonged boiling increased the DNA concentration but decreased the intactness of DNA. (3) Ginseng could be differentiated from American ginseng by multiplex PCR. (4) Individual herbs in the decoctions with prolonged boiling time were identified by amplifying short DNA fragments. We also demonstrated the presence of DNA in a number of animal and plant medicinal granules and they could be amplified by species-specific primers.

Identification of constituent herbs in ginseng decoctions by DNA markers.

BackgroundDNA in herbal decoctions is usually fragmented by extensive boiling and is usually regarded as unsuitable for molecular authentication. This study aims to evaluate the feasibility for molecular authentication methods by multiplex polymerase chain reaction (PCR) and DNA sequencing for decoctions.MethodsThe DNA extraction procedure, sample pulverization and boiling time were examined in (1) single herb decoction with Panax ginseng (“ginseng”) or P. quinquefolius (“American ginseng”), (2) decoctions of two classical ginseng prescriptions of five herbal materials (Aconitum carmichaeli, Atractylodes macrocephala, P. ginseng, Glycyrrhiza uralensis and Zingiber officinale) and (3) a commercial ready-to-serve ginseng soup. Primers were designed from 26S-18S, ITS2 and trnH-psbA region, with DNA sequences obtained from GenBank. Multiplex PCR was also employed in ginseng or American ginseng decoctions to differentiate between these two herbs.ResultsAll six herbal species tested in this study could be identified in decoctions. We had four main observations: (1) sample pulverization before boiling improved PCR detection; (2) prolonged boiling increased the DNA concentration but decreased the intactness of DNA fragments; (3) ginseng could be differentiated from American ginseng by multiplex PCR; (4) identification of individual herbs in multi-herb decoctions with prolonged boiling time of 180 min could be achieved.ConclusionsDNA could be amplified from extensively boiled ginseng decoctions, multi-herb decoctions and commercial soup although DNA degradation was critical to successful PCR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0029-x) contains supplementary material, which is available to authorized users.

Concentration and Distribution of Nuclei and Plastids in Xylem Cells in Cunninghamia lanceolata and Aquilaria sinensis

After programmed cell death (PCD), heartwood formation, storage, and processing, wood DNA degradation occurs to varying degrees. The concentration and distribution of nuclei and plastids in xylem cells of Cunninghamia lanceolata and Aquilaria sinensis, treated under different conditions of processing and storing, were studied by analyzing the distribution frequency, area, and signal intensity, in specimens that had been stained with aceto-carmine, DAPI, and I2-KI. Most of the nuclei and plastids were present in the ray cells, and a small quantity of nuclei and plastids were present in the axial parenchyma cells. There was an indication that the concentration of the remaining nuclei and plastids in the xylem cells was mainly affected by the xylem heartwood formation, storage time, and temperature. The nuclei and plastids content of the sapwood was greater than that of the heartwood. However, the nuclei and plastids content of the fresh wood was greater than that of the processed and stored wood. An estimation of the quantity of nuclei and plastids using staining methods could provide a direct basis for the appropriate selection of a procedure for DNA extraction.

Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing®.

One of the major reasons for pregnant women to ask for prenatal diagnosis is to detect fetal chromosomal aneuploidies. Analysis of allele ratios of SNPs has been used for prenatal detection of fetal aneuploidies using MALDI-TOF mass spectrometry (MS). However, quantitative SNP genotyping by MALDI-TOF MS is challenging. To obtain a better quantification of allelic ratios, a Pyrosequencing(®) protocol for SNP genotyping has been developed to perform prenatal diagnosis of aneuploidies.To avoid the laborious process and risk of cross-contamination brought in by DNA extraction procedures, a PCR assay, which can amplify DNA directly from cells in amniotic fluid, has been developed. Pre-amplification steps such as cell enrichment and heating are required to obtain sufficient amounts of amplification products.In this chapter, SNPs on chromosome 21 are used to detect trisomy 21 as an example of aneuploidy by quantifying the allele ratio using Pyrosequencing. Primer selection for PCRs and Pyrosequencing reactions, optimization of nucleotide dispensation orders, establishment of cutoff values for trisomy 21, and interpretation of data are all factors essential for a successful diagnosis and are discussed in detail herein.

Real-Time Polymerase Chain Reaction Assay for the Diagnosis of Pulmonary Tuberculosis.

To the editor: The recent report on Real-time polymerase chain reaction (PCR) assay for the diagnosis of pulmonary is very interesting1. Kim et al.1 noted that Real-time PCR from selective bronchoscopic aspirates enhances the diagnostic yield much more when added to sputum examination. In fact, polymerase chain reaction assay for tuberculosis (TB) is considered new and useful diagnostic test. However, there are some concerns on this assay. First, false-positive of the test can be seen in cases with treated or old lesion from pulmonary TB2. Second, the low sensitivity of the test can be seen1,2,3. In fact, there are many possible causes of low sensitivity including to type of specimen transfer and duration time, the DNA extraction procedure, DNA amplified process, DNA extraction, and reproducible technique2. Finally, the cost effectiveness of the test is the topic for further studied.

The hidden ‘mycobacteriome’ of the human healthy oral cavity and upper respiratory tract

The incidence of opportunistic non-tuberculous mycobacteria (NTM) infections has increased considerably in the past decades causing an array of infections, including respiratory and soft-tissue infections. NTM are ubiquitous and can be found in numerous environments, including households and water plants. However, NTM have not been reported to be associated with the healthy human oral microbiome. Since the oral cavity and upper respiratory track are the main ports of entry of microorganisms into the human body, elucidating NTM diversity and prevalence will assist in the assessment of the potential risks of infection elicited by these opportunistic pathogens. Here, we report the identification of a ‘non-tuberculous mycobacteriome’ in healthy individuals. We employed a modified DNA extraction procedure in conjunction with mycobacterial-specific primers to screen niches in the oral cavity (buccal mucosa and dental plaque) and upper respiratory tract (nostrils and oropharynx) of 10 healthy subjects. A total of 50 prevalent operational taxonomic units sequenced on MiSeq (Illumina) using 16S rRNA V3–V4 region were detected across all screened niches, showing the presence of diverse NTM communities. NTM DNA was detected in the nostrils of all 10 subjects, in buccal mucosa of 8 subjects, in the oropharynx of 7 subjects, and in the dental plaques of 5 subjects. Results from quantitative PCR showed each individual harbored 103–104 predicted NTM per each screened niche. The modification of standard DNA isolation methods to increase sensitivity toward mycobacterial species represents an important step to advance the knowledge of the oral as well as the overall human microbiome. These findings clearly reveal for the first time that healthy individuals harbor a ‘non-tuberculous mycobacteriome’ in their oral cavity and upper respiratory tract and may have important implications in our understanding of infections caused by NTM.

Prevalence of human papillomavirus in esophageal carcinoma in Tangshan, China.

To study the prevalence of human papillomavirus (HPV) in esophageal carcinoma in Tangshan, China, a high-incidence area. Formalin-fixed, paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan. DNA was extracted from all 198 specimens to detect HPV by polymerase chain reaction (PCR). β-globin PCR was performed to check the quality of the DNA extraction procedure. PCR was performed to detect a wide range of HPV types, and type-specific PCR was performed to detect HPV types 16 and 18. Negative and positive controls were used for HPV 16 and 18 detection. The DNA extraction method in this study appeared to be more effective than other previously reported methods. After DNA extraction, more than 98% of the tissue specimens had an acceptable result in the DNA qualification test (β-globin PCR). The overall prevalence of HPV in tumor tissues by GP6+/GP5+ PCR was 79.79%, and the prevalence of HPV types 16 and 18 was 40.40% and 47.47%, respectively. PCR demonstrated the presence of HPV, and direct sequencing confirmed the HPV genotypes. All HPV-positive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the Basic Local Alignment Search Tool database to evaluate the HPV types. This analysis confirmed the presence of HPV types 16 and 18. DNA of high-risk HPV types 16 and 18 is present in esophageal tumors, implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: The SPIDIA experience

BackgroundDNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. Methods157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. Results/conclusionsOur results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.