Direct qPCR Research Articles

Impact of PCR-based molecular analysis in daily diagnosis for the patient with gliomas

The WHO2016 CNS update requires a combined histological and molecular assessment. To assess the major aberrations such as co-deletion of complete chromosome arms 1p and 19q (Co-del), isocitrate dehydrogenase and histone H3 mutations, direct sequencing, multiplex ligation-dependent probe amplification and/or FISH are methods considered to be "golden standard" in the community. However, these methods are expensive and complicated. The aim of this study is verification of the sensitivity of the simple PCR-based techniques for assessment of molecular information in daily diagnosis. We analyzed a total number of 80 patients with gliomas. FISH and PCR-based microsatellite analysis were compared for Co-del assessment. Direct sequencing and qPCR using hig-resolution melting (HRM) were compared for IDH and histone H3 mutations. The sensitivity and specificity of FISH were 0.71 and 0.79, respectively. FISH using a commercially available Vysis probe had a risk of high false-positive rate (0.25). For assessment of IDH1 mutations, the sensitivity and specificity of HRM were 1.0 and 0.96, respectively. For assessment of IDH2 and H3 mutations by HRM, both sensitivity and specificity were 1.0. We consider PCR-based molecular analysis to be a simple and accurate technique in daily diagnosis that is readily available for a small scientific facility.

221 - Validation of a hybrid qPCR-based mycoplasma test method

A sensitive, robust, and rapid in-process mycoplasma test method was developed to provide a quicker turnaround time compared to the traditional culture based methods without compromising quality. This hybrid qPCR-based method was specifically designed to detect down to 10 CFU/mL for most species tested, accommodate a wide variety of sample types, generate accurate results in 3 days, and combat false negative as well as false positive results. A successful validation was conducted in alignment with the qualitative assay parameters recommended in EP 2.6.7 and PDA Technical Reports 33 and 50, establishing comparability to the compendial method. This presentation will highlight a risk based approach inclusive of technical and regulatory considerations for intended use. Discussion will focus on the merits of a hybrid vs direct qPCR method for both in-process and final release applications.

An integrated cell absorption process and quantitative PCR assay for the detection of the infectious virus in water

Here we developed an integrated cell absorption process and quantitative (reverse transcription) polymerase chain reaction (ICAP-q(RT)PCR) assay to detect infectious viruses, which based on the detection of the viral nucleic acid (RNA or DNA) in the early stage of viral attachment and entry towards cells. The results showed that the poliovirus or adenovirus whose concentration was as low as 0.2 TCID50/mL could be detected by ICAP-q(RT)PCR after 4 h incubation. The ICAP-q(RT)PCR exhibited much higher sensitivity than the plaque assay. In parallel, it took shorter time to detect the viruses towards field samples compared with the integrated cell culture (ICC)-qPCR, but could still get the consistent detecting results with ICC-qPCR. This method is verified by detecting four different kinds of viruses including poliovirus, adenovirus, rotavirus, and astrovirus, which existed in the actual water samples. Among all the 24 Jinhe river samples, 50% (12/24) of river water samples were positive for poliovirus when detected by ICAP-q(RT)PCR, which was in accordance with the results detected by ICC-qPCR. However, 21% (5/24) and 68% (18/24) of the samples were detected to be positive for poliovirus by the plaque counting and the direct qPCR method, respectively. Compared with ICAP-q(PT)PCR and ICC-qPCR, the detecting results of qPCR or plaque assay displayed a marked expansion or decline, respectively, which lead to the evident deviations in the accuracy. The results demonstrated that our developed ICAP-q(RT)PCR method could dramatically reduce the test duration and quite improve the sensitivity towards infectious viruses. Therefore, the ICAP-q(RT)PCR method could be an effective and quantitative tool for detecting infectious viruses in water environments.

Improvements in Tritrichomonas foetus molecular testing.

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

Improved sample preparation for direct quantitative detection of Escherichia coli O157 in soil using qPCR without pre-enrichment.

SummaryThe prominence of fresh produce as a vehicle for foodborne pathogens such as enterohaemorrhagic Escherichia coli (EHEC) O157 is rising, where disease cases can cause hospitalization and in some cases death. This rise emphasises the necessity for accurate and sensitive methods for detection of pathogens in soil, potential sources of contamination of fresh produce. The complexity of the soil matrix has previously proven prohibitive to pathogen detection via molecular methods without the use of a culture enrichment step, thereby excluding the detection of viable but non‐culturable cells. Here, a sample preparation procedure to facilitate a direct qPCR assay is developed for the detection of E. coli O157 in soil, bypassing culture steps in favour of sample separation through pulsification release and filtration. In sand and peat‐based compost, the method is sensitive to 10 CFU g−1 soil. When testing soils from agricultural sites, it was found that several were qPCR positive for E. coli O157 while being culture‐negative, with peat‐based compost possessing a concentration of 200 tir gene copies per gram. This procedure offers a rapid, quantitative assessment of the potential presence of E. coli O157 in soils which can act as a prescreen of their suitability to grow fresh produce safely.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients’ sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay.The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28μg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6–3.1μg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32–1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests.Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

Comparison of methods to evaluate the fungal biomass in heating, ventilation, and air-conditioning (HVAC) dust

Heating, ventilation, and air-conditioning (HVAC) systems contain dust that can be contaminated with fungal spores (molds), which may have harmful effects on the respiratory health of the occupants of a building. HVAC cleaning is often based on visual inspection of the quantity of dust, without taking the mold content into account. The purpose of this study is to propose a method to estimate fungal contamination of dust in HVAC systems. Comparisons of different analytical methods were carried out on dust deposited in a controlled-atmosphere exposure chamber. Sixty samples were analyzed using four methods: culture, direct microscopic spore count (DMSC), β-N-acetylhexosaminidase (NAHA) dosing and qPCR. For each method, the limit of detection, replicability, and repeatability were assessed. The Pearson correlation coefficients between the methods were also evaluated. Depending on the analytical method, mean spore concentrations per 100cm2 of dust ranged from 10,000 to 682,000. Limits of detection varied from 120 to 217,000 spores/100cm2. Replicability and repeatability were between 1 and 15%. Pearson correlation coefficients varied from -0.217 to 0.83. The 18S qPCR showed the best sensitivity and precision, as well as the best correlation with the culture method. PCR targets only molds, and a total count of fungal DNA is obtained. Among the methods, mold DNA amplification by qPCR is the method suggested for estimating the fungal content found in dust of HVAC systems.

Risk of Illness with Salmonella due to Consumption of Raw Unwashed Vegetables Irrigated with Water from the Bogotá River.

The Bogotá River receives untreated wastewater from the city of Bogotá and many other towns. Downstream from Bogotá, water from the river is used for irrigation of crops. Concentrations of indicator organisms in the river are high, which is consistent with fecal contamination. To investigate the probability of illness due to exposure to enteric pathogens from the river, specifically Salmonella, we took water samples from the Bogotá River at six sampling locations in an area where untreated water from the river is used for irrigation of lettuce, broccoli, and cabbage. Salmonella concentrations were quantified by direct isolation and qPCR. Concentrations differed, depending on the quantification technique used, ranging between 107.7 and 109.9 number of copies of gene invA per L and 105.3 and 108.4 CFU/L, for qPCR and direct isolation, respectively. A quantitative microbial risk assessment model that estimates the daily risk of illness with Salmonella resulting from consuming raw unwashed vegetables irrigated with water from the Bogotá River was constructed using the Salmonella concentration data. The daily probability of illness from eating raw unwashed vegetables ranged between 0.62 and 0.85, 0.64 and 0.86, and 0.64 and 0.85 based on concentrations estimated by qPCR (0.47-0.85, 0.47-0.86, and 0.41-0.85 based on concentrations estimated by direct isolation) for lettuce, cabbage, and broccoli, respectively, which are all above the commonly propounded benchmark of 10-4 per year. Results obtained in this study highlight the necessity for appropriate wastewater treatment in the region, and emphasize the importance of postharvest practices, such as washing, disinfecting, and cooking.

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

Density Distribution of Pharyngeal Carriage of Meningococcus in Healthy Young Adults: New Approaches to Studying the Epidemiology of Colonization and Vaccine Indirect Effects.

Improved understanding of Neisseria meningitidis (Nm) carriage biology and better methods for detection and quantification would facilitate studies of potential impact of new vaccines on colonization and transmission in adolescents. We performed plate cultures on 107 oropharyngeal swabs stored frozen in skim milk tryptone glucose glycerol (STGG) broth and previously positive for Nm. We compared quantitative polymerase chain reaction (qPCR) detection of Nm in 601 STGG-swabs with culture. Using qPCR (n = 87), a log-phase broth culture standard curve and semiquantitative plate cultures (n = 68), we measured density of carriage. We compared qPCR genogrouping of DNA extracts from STGG-swabs and from plate culture lawns (n = 110) with purified isolates (n = 80). Swab storage resulted in only 10% loss of culture sensitivity. Direct sodC qPCR Nm detection yielded more positives (87/601, 14.5%) than culture (80/601, 13.3%). Most samples (57/110) positive by culture were also positive by qPCR and vice versa, but discrepancies (single positives) were frequent among low-density samples. sodC qPCR was positive in 79/80 isolates but in only 65 by ctrA qPCR. Density both by culture and qPCR varied across 4 orders of magnitude with the majority being low (<50 bacteria-gene copies/mL) and a minority being high (>1000). Genogrouping qPCRs yielded more positive results when performed on DNA extracts from lawn cultures. We provide the first description of the distribution of Nm carriage density. This could be important for understanding transmission dynamics and population-level effectiveness of adolescent vaccine programs. Storage of swabs frozen in STGG for batched laboratory analysis facilitates carriage studies and direct sodC qPCR for Nm combined with qPCR genogrouping of lawn culture extracts provides accurate, detailed description of colonization.

High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells

Chlamydiae are obligate intracellular bacteria developing in an intracytoplasmic niche, the inclusion. Chlamydia growth measurement by inclusion counting is a key task in the development of novel antichlamydial antibiotics and in vaccine studies. Most of the current counting methods rely on the immunofluorescent staining of the inclusions and either manual or automatic microscopy detection and enumeration. The manual method is highly labor intensive, while the automatic methods are either medium-throughput or require automatic microscopy. The sensitive and specific PCR technology could be an effective method for growth related chlamydial DNA detection; however the currently described PCR approaches have a major limitation, the requirement of purification of DNA or RNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of Chlamydia trachomatis DNA directly from the infected HeLa cells. With our method we were able to detect the bacterial growth in a 4 log scale (multiplicity of infection (MOI): 64 to 0.0039), with high correlation between the biological and technical replicates. As a further proof of the method, we applied the direct qPCR for antibiotic minimum inhibitory concentration (MIC) measurements. The measured MICs of moxifloxacin, tetracycline, clarithromycin and compound PCC00213 were 0.031μg/ml, 0.031μg/ml, 0.0039μg/ml and 6.2μg/ml respectively, identical or close to the already published MIC values. Our direct qPCR method for chlamydial growth and antibiotic MIC determination is less time-consuming, more objective and more sensitive than the currently applied manual or automatic fluorescent microscopy- based methods.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013–March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System®). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170–230 MAP cells/g (62–115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells

Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named ‘direct resDNA qPCR’, the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods.

Detection of small amounts of human adenoviruses in stools: comparison of a new immuno real-time PCR assay with classical tools

The detection of low virus concentrations in biological matrices, especially stool samples, is facing significant limitations as far as common diagnostic methods (enzyme-linked-immunosorbent assay (ELISA) or quantitative real-time PCR (qPCR)) are considered. Here the development of a new immuno real-time PCR (iPCR) is described and its performance in the detection of human adenoviruses (HAdVs) in spiked stools is compared with those of ELISA and qPCR assays. For the iPCR, detection of the sandwich formed by the complexation of capture antibody-antigen-detection antibody was performed by qPCR thanks to the substitution of peroxydase by a chimeric DNA. This modification increased the detection sensitivity 200-fold compared to ELISA. The direct qPCR results revealed that only 0.3–9.5% of the spiked HAdV were detectable, resulting from important losses of DNA occurring at the extraction step. This step was not necessary in the iPCR workflow, avoiding this drawback. The losses of viral particles occurred at the elution step from the stool only. The recovery rate of the iPCR was thus better and ranged between 21 and 54%. As a result, iPCR enabled the detection of lower virus concentrations in stool samples compared to those detected by ELISA and qPCR. The iPCR could be considered as a ‘hyper sensitive ELISA’ for early detection of HAdV infections, especially in the case of immunocompromised patients after haematopoietic stem cell transplant.

The association of detection method, season, and lactation stage on identification of fecal shedding in Mycobacterium avium ssp. paratuberculosis infectious dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative organism of Johne’s disease. Although fecal culture is considered the standard diagnostic test, the long incubation times, costs, and intermittent shedding of MAP hinder efficient screening programs based on culture results. The primary objectives of this study were to determine the detection ability of solid culture, broth culture, and real-time PCR (qPCR) for MAP in fecal samples and to assess how shedding patterns of MAP in feces vary with lactation stage and season. This knowledge could improve the use of these diagnostic assays in Johne’s management programs. For this study, 51 MAP-infectious cows from 7 Atlantic Canadian dairy farms had fecal samples collected monthly over a 12-mo period. Samples were analyzed for MAP bacterial load via solid culture, broth culture, and qPCR. For all fecal samples, 46% [95% confidence interval (CI): 40 to 51%] were positive by solid culture, 55% (95% CI: 50 to 60%) by broth culture, and 78% (95% CI: 73 to 82%) by qPCR. Sensitivity of qPCR was numerically higher in the dry and postpartum lactation periods, and qPCR detection in summer and fall was 85% of that in winter and spring. Furthermore, culture-determined moderate or light shedding categories generally corresponded to qPCR cycle threshold values <35, but heavy shedding categories corresponded to qPCR values <29. Direct fecal qPCR is a MAP detection method that is quick and less costly than culture techniques, and it avoids the use of decontamination steps that could decrease numbers of bacteria in a sample below the detection limit. This study indicates that, for known MAP-positive cows, fecal qPCR had high sensitivity of MAP detection, thereby supporting the use of direct fecal qPCR as part of a Johne’s herd control program.

Abstract 4897: Genetic variants and expression of AEG-1 in relation to clinical outcome and radiotherapy response in colorectal cancer patients and cell lines

Abstract Background: Colorectal cancer (CRC) is the third most common cancer in men and the second in woman worldwide. The therapy of CRC has been critically improved during the past two decades, but the treatment response varies significantly between different treatments and patients. Therefore it is of need to search for biomarkers for more suitable prognosis and treatment. The aim of this study was to investigate genetic variants of the astrocyte elevated gene-1 (AEG-1), its expression in CRC patient samples and colon cancer cell lines and the potential correlation to clinicopathological variables and treatment response. Materials and Methods: We analysed genetic variants and the mRNA and protein expression of AEG-1 in 593 CRC patient samples by direct Sanger sequencing, qPCR and immunohistochemistry. Of the patients, 158 rectal cancer patients were enrolled in the Swedish clinical trial of preoperative radiotherapy (RT). AEG-1 expression in response to γ-irradiation was analysed by Western blot in 5 colon cancer cell lines. We inhibited the AEG-1 expression by siRNA in the cell lines, and their survival was analysed after γ-irradiation. Results: By direct sequencing we found 29 variants, of which 12 were novel. Six of the variants were exonic and 23 intronic. The variant c.1353G&amp;gt;A, rs2331652 was found only in 4 samples and the variant c.1679-6 T&amp;gt;C in only one sample. In the cell lines KM12C, KM12SM and KM12L4a we found a deletion in exon 4 (c.595_598delAGAG). AEG-1 mRNA and protein expression revealed a significant increased AEG-1 expression in the primary tumors and metastases compared to the normal mucosa in all patient cohorts (p&amp;lt;0.006) and on the mRNA level a higher expression in tumors located in the rectum compared to tumors in the colon (p=0.047). In the rectal cancer patients from the Swedish trail of preoperative RT, a high AEG-1 expression correlated with a higher risk of distant recurrence and disease free survival (p=0.001 respectively) independently of the tumor stage, only in patients receiving preoperative RT. AEG-1 knock-down in the radioresistant cell lines KM12L4a, SW480 and SW620 resulted in reduction of the survival after radiation, but not in the radiosensitive cell lines KM12C and HCT116. The AEG-1 expression was up-regulated shortly after 2 Gy γ-radiation in KM12C, KM12L4a and SW480, but not in SW620 and HCT116. Conclusion: We found several novel AEG-1 variants. The AEG-1 expression at the mRNA and/or protein level was up-regulated in the primary tumour and metastases compared to the normal mucosa. Furthermore we showed for the first time that the AEG-1 expression is an independent prognostic factor for distant recurrence and disease-free survival in rectal cancer patients after treatment with preoperative RT. The cell line studies suggest AEG-1 as a promising radiosensitizing target for rectal cancer. Citation Format: Sebastian Gnosa, Veronika Patcha Brodin, Ivana Ticha, Gunnar Adell, Gunnar Arbman, Hong Zhang, Xiao-Feng Sun. Genetic variants and expression of AEG-1 in relation to clinical outcome and radiotherapy response in colorectal cancer patients and cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4897. doi:10.1158/1538-7445.AM2014-4897

Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins

Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a “direct qPCR” approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA (“extraction qPCR”). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1–0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.

Direct qPCR quantification of unprocessed forensic casework samples

The current short tandem repeat (STR) typing workflow for forensic casework samples involves sample collection, sample screening, DNA extraction, DNA qPCR quantification and STR amplification. Although very effective and powerful, this workflow still has room for improvements. For example, the screening assays in practice do not provide DNA related information and also do not work with touch DNA samples, which make up of the majority of the property crime samples. It is known that not all DNA samples have equal probative values. Considering the DNA backlog situation crime laboratories face today, an effective screening tool would be highly desirable. It would allow forensic scientists to prioritize the DNA samples so that the limited resources would be first spent on samples that would have better chances of producing informative STR profiles. qPCR assay does provide DNA quantity and gender information and would be an ideal screening tool. However, prior to quantification, sample extraction and purification are required. By the time a DNA sample is ready for qPCR quantification, time and resources have already been spent on samples that should have been given low priority or excluded from further processing if DNA quantity and gender information were known. To overcome this problem, a direct quantification technology is developed to allow qPCR quantification of casework samples without the need for DNA extraction and purification. The key to a direct qPCR assay is the PE-Swab, a novel sample collection device. A small sample punch can be generated from a PE-Swab and placed in a qPCR reaction for quantification. After optimizing the punch size and the quantification software baseline setting, accurate DNA quantification can be obtained from a sample without the need to carry out DNA extraction and purification. Proof of concept studies were done with low lever touch samples as well as blood samples. The PE-Swab also allows direct STR amplification of casework samples without the need for DNA extraction. Besides its potential as a screening tool, the direct qPCR assay can also be used to normalize the DNA input for a direct STR amplification reaction. The feasibility of the direct qPCR/direct STR amplification workflow was demonstrated with touch DNA samples and blood stain samples.

Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

Quantification of E. coli O157 and STEC in feces of farm animals using direct multiplex real time PCR (qPCR) and a modified most probable number assay comprised of immunomagnetic bead separation and qPCR detection

To better understand Escherichia coli O157:H7 on-farm transmission dynamics requires sensitive methods for quantification of a broad range of concentrations of target organisms. For this purpose, a multiplex real time PCR (qPCR) assay was developed for quantification of O157 E. coli from 1g fecal samples of cattle and other animal species, targeting the Shiga toxin genes (stx1 and stx2) and the O157 somatic antigen gene, per. The multiplex qPCR assay provided specific detection across a broad range of bacterial concentrations with a lower limit of detection (LOD) of 101 genome copies which is equivalent to 101 bacteria. However, the LOD, when direct qPCR was applied to quantification of the targets in the feces of dairy cattle, was 103 genome copies per gram of feces. Enumeration below the threshold for direct qPCR was performed using a modified most probable number (mMPN) method whereby E. coli O157 in enriched samples was isolated using immunomagnetic bead separation (IMS) and detected using qPCR, thus reducing the time and logistic constraints of biochemical/serological/gel analysis. Application of the mMPN (IMS/qPCR) assay to samples that were negative when tested using direct qPCR alone permitted quantification of low levels of E. coli O157 below levels detectable with direct qPCR. The direct qPCR and mMPN (IMS/qPCR) assays were applied to fecal samples from dairy, beef, swine and poultry feces. This approach can be employed to gain a better understanding of the patterns of infection in animals for analysis of on-farm transmission dynamics, for evaluating the effects of on-farm control strategies and for risk assessment in public health.