Magnetic Capture Hybridization Research Articles

Global distribution, traditional and modern detection, diagnostic, and management approaches of Rhizoctonia solani associated with legume crops.

Sustainable development relies heavily on a food system that is both safe and secure. Several approaches may lead to sustainability and food safety. An increase in the cultivation of legume crops is one of the approaches for enhancing agricultural viability and ensuring adequate food supply. Legumes may increase daily intake of fiber, folate, and protein as substitutes for meat and dairy. They are also crucial in various intercropping systems worldwide. However, legume production has been hampered by Rhizoctonia solani due to its destructive lifestyle. R. solani causes blights, damping off, and rotting diseases in legume crops. Our knowledge of the global distribution of R. solani associated with legume crops (alfalfa, soybean, chickpea, pea, lentil, common bean, and peanut), detection, diagnosis, and management of legume crops diseases caused by R. solani is limited. Traditional approaches rely on the incubation of R. solani, visual examination of symptoms on host legume crops, and microscopy identification. However, these approaches are time-consuming, require technical expertise, fail to detect a minimal amount of inoculum, and are unreliable. Biochemical and molecular-based approaches have been used with great success recently because of their excellent sensitivity and specificity. Along with conventional PCR, nested PCR, multiplex PCR, real-time PCR, magnetic-capture hybridization PCR, and loop-mediated isothermal amplification have been widely used to detect and diagnose R. solani. In the future, Next-generation sequencing will likely be used to a greater extent to detect R. solani. This review outlines global distribution, survival, infection and disease cycle, traditional, biochemical, molecular, and next-generation sequencing detection and diagnostic approaches, and an overview of the resistant resources and other management strategies to cope with R. solani.

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review.

Phytopathogenic fungal species can cause enormous losses in quantity and quality of crop yields and this is a major economic issue in the global agricultural sector. Precise and rapid detection and identification of plant infecting fungi are essential to facilitate effective management of disease. DNA-based methods have become popular methods for accurate plant disease diagnostics. Recent developments in standard and variant polymerase chain reaction (PCR) assays including nested, multiplex, quantitative, bio and magnetic-capture hybridization PCR techniques, post and isothermal amplification methods, DNA and RNA based probe development, and next-generation sequencing provide novel tools in molecular diagnostics in fungal detection and differentiation fields. These molecular based detection techniques are effective in detecting symptomatic and asymptomatic diseases of both culturable and unculturable fungal pathogens in sole and co-infections. Even though the molecular diagnostic approaches have expanded substantially in the recent past, there is a long way to go in the development and application of molecular diagnostics in plant diseases. Molecular techniques used in plant disease diagnostics need to be more reliable, faster, and easier than conventional methods. Now the challenges are with scientists to develop practical techniques to be used for molecular diagnostics of plant diseases. Recent advancement in the improvement and application of molecular methods for diagnosing the widespread and emerging plant pathogenic fungi are discussed in this review.

Quantatitative assays for covalently closed circular DNA of hepatitis B virus

<p indent=0mm>Chronic infection of hepatitis B virus (HBV) is still one of the public health concerns affecting human health. Although the prophylactic HBV vaccines was introduced over three decades ago, the total number of chronic hepatitis B (CHB) infection still accounts for 3.5% (257 million) of the world’s population, with approximately 800000 annual deaths related to the viral infection. Currently, drugs including PEG-IFN and NAs are widely used in the treatment of HBV infection with certain degree of success. It is still difficult to achieve a high proportion of “clinical cure” with existing treatment methods. The presence of covalently closed circular DNA (cccDNA) in the infected hepatocytes is the underlying mechanism for the long-term HBV persistence and is also essential for off-therapy viral relapse. The current treatments have no or limited effects on the suppression of intrahepatic cccDNA. The availability for a reliable cccDNA quantification assay would facilitate the developmentof anti-viral drugs targeting cccDNA-targeting. In this review, we summarized the advantages and disadvantages of the existing methods for cccDNA quantitation. Specifically, the extraction method of Hirt DNA combined with Exonuclease V can effectively remove other forms of HBV DNA, and suitable for cccDNA extraction, but it needs to be further optimized. Southern blot is considered as the “gold-standard” method for cccDNA detection, but it is less sensitive and requires cumbersome procedures, making it an unlikely method for high-throughput screening used for drug development or for clinical applications. The PCR-based methods are more convenient and sensitive for cccDNA detection. However, the reliability of PCR-based method requires further optimization and validation. Different PCR-based methods were developed for quantitation of HBV cccDNA. They include selective qPCR, droplet-digital PCR, rolling circle amplification PCR,<italic> in situ</italic> PCR, invader assay, magnetic capture hybridization qPCR, etc. These PCR-based methods are widely used in clinical and laboratory research for quantitation of cccDNA. Efforts need to be made to minimize the interference from other forms of DNAs in the assays. More recently, hepatitis B core related antigen (HBcrAg)was found to be a novel marker of HBV, and its level in serum is highly correlated with intracellular cccDNA level. This biomarker, the HBcrAg level, can be used to indicate the suppression of cccDNA during different clinical antiviral therapies. The correlation needs further analysis with additional data sets. In summary, we present a review on the quantitation assays for HBV cccDNA. Such assays are important for drug developments and clinical applications for improved management of HBV infection related diseases. Performance and characteristics of various methods were compared with the areas of improvements for different assays.

Current PCR-based methods for the detection of mycotoxigenic fungi in complex food and feed matrices

Mycotoxins are toxic secondary fungal metabolites produced by certain types of filamentous fungi, such asAspergillus, Fusarium, andPenicillium spp. Mycotoxigenic fungi and their produced mycotoxins are considered to be an important issue in food and feed safety due to their toxic effects like carcinogenicity, immunosuppression, neurotoxicity, nephrotoxicity, and hepatotoxicity on humans and animals. To boost the safety level of food and feedstuff, detection and identification of toxins are essential at critical control points across food and feed chains. Zero-tolerance policies by the European Union and other organizations about the extreme low level of tolerance of mycotoxins contamination in food and feed matrices have led to an increasing interest to design more sensitive, specific, rapid, cost-effective, and safer to use mycotoxigenic fungi detection technologies. Hence, many mycotoxigenic fungi detection technologies have been applied to measure and control toxins contamination in food and feed substrates. PCR-based mycotoxigenic fungi detection technologies, such as conventional PCR, real-time PCR, nested PCR, reverse transcriptase (RT)-PCR, loop-mediated isothermal amplification (LAMP),in situ PCR, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR DGGE), co-operational PCR, multiplex PCR, DNA arrays, magnetic capture-hybridization (MCH)-PCR and restriction fragment length polymorphism (RFLP), would contribute to our understanding about different mycotoxigenic fungi detection approaches and will enhance our capability about mycotoxigenic fungi identification, isolation and characterization at critical control points across food and feed chains. We have assessed the principles, results, the limit of detection, and application of these PCR-based detection technologies to alleviate mycotoxins contamination problem in complex food and feed substrates. The potential application of these detection technologies can reduce mycotoxins in complex food and feed matrices.

Elimination of the Crown Gall Pathogen, Agrobacterium vitis, from Systemically Infected Grapevines by Tissue Culture

Agrobacterium vitis causes crown gall, a serious disease of grapevines that is common in grape growing regions worldwide. Tissue culture approaches were previously tested for elimination of the bacterium from grapevines, but the detection methods for assessing their effectiveness were much less sensitive than the recently developed magnetic capture hybridization real-time PCR. The latter method was applied in this study to evaluate the elimination of A. vitis from cuttings propagated from severely crown gall-diseased Vitis vinifera cv. Riesling vines by dissecting and culturing apical and axillary buds, as well as meristems, from shoot tips and micropropagating them in tissue culture to recover plantlets. Although the distribution of A. vitis in infected mother vines varied greatly, apparently bacteria-free meristematic tissues were successfully used for the establishment of vines for which bacterial populations were undetectable, even after a dormancy period. It is anticipated that the production and use of clean grapevines in vineyards will delay the early onset of crown gall and contribute to vineyard productivity.

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10CFU in samples with individual pathogens and 102CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Identifying Environmental Sources of Agrobacterium vitis in Vineyards and Wild Grapevines

Agrobacterium vitis, the primary cause of grape crown gall disease, is known to survive internally in grapevines and to spread in propagation material. In this study, we showed that the bacterium can be detected in dormant grape buds and on surfaces of leaves collected from commercial vineyards. Using a highly selective and sensitive method based on magnetic capture hybridization (MCH) together with real-time PCR, we detected A. vitis in as much as 90% of dormant bud samples and in up to 40% of leaf samples from individual vineyards. The highest percentages of detection occurred in samples collected from vineyards with high incidences of crown gall. A. vitis was also detected in 22% of wild grapevines (Vitis riparia) collected in New York and in 25% of feral grapevines that included V. californica in California. Several of these vines were growing more than 2 km from commercial vineyards, demonstrating that wild grapevines can serve as a significant inoculum reservoir. The specificity of the MCH and real-time PCR assay used to detect tumorigenic A. vitis in the environment was further demonstrated by the finding that 69 nontumorigenic strains from regions across the United States did not amplify a virD2 PCR product.

Distribution of Agrobacterium vitis in Grapevines and Its Relevance to Pathogen Elimination.

Agrobacterium vitis, the cause of crown gall disease on grapevine, survives internally in vines and can be spread in cuttings for propagation. The possibility of generating pathogen-free vines through tissue culture makes it essential to understand the distribution of the pathogen in grapevines. A highly sensitive magnetic capture hybridization procedure along with real-time polymerase chain reaction were used to measure the distribution of tumorigenic A. vitis in dormant canes and green shoots of grapevines. Tumorigenic A. vitis was distributed from the basal to apical nodal and internodal tissues of canes as well as in nonlignified green shoots. In experiments conducted in 2013, A. vitis was detected in up to 17% of shoot tips and 52% of meristems of greenhouse-grown plants initiated from known A. vitis-contaminated cuttings. A lower frequency of detection was observed from surface-disinfected shoot tips (7%) as compared with nondisinfected tips (37%), suggesting epiphytic survival on green tissues. In 2014, vines propagated from cuttings collected from crown gall-infected vines from a different vineyard yielded lower incidences of A. vitis from shoot tips, and the bacterium was not detected in meristems. Tumorigenic A. vitis was also detected in cuttings of wild grapevines (Vitis riparia) that were collected both adjacent to and far removed from commercial vineyards.

Development of magnetic capture hybridization and quantitative polymerase chain reaction for hepatitis B virus covalently closed circular DNA.

Background:Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available.Objectives:Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology.Materials and Methods:The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B.Results:Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 102-106 IU/mL was revealed (r2 = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA.Conclusions:The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.

Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of TumorigenicAgrobacterium vitisin Grapevines

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 10(1) CFU/ml compared with 10(5) CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 10(4) CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

Progress Towards a Real‐time PCR Assay for the Simultaneous Detection of Clavibacter michiganensis subsp. michiganensis and Pepino mosaic virus in Tomato Seed

AbstractQuarantined phytopathogens such as Pepino mosaic virus (PepMV) and Clavibacter michiganensis subsp. michiganensis can be introduced into tomato transplant houses and fields on infested seed and thereby cause significant economic losses. Hence, specific and sensitive seed health assays are required to exclude these organisms. Currently, separate assays must be conducted on seed samples for each pathogen, making the process time‐consuming and expensive. One approach to improve the efficiency of seed health testing is multiplex real‐time PCR; however, PCR can be inhibited by compounds co‐extracted from seed tissues with nucleic acids. To address this concern, we explored the use of magnetic capture hybridization (MCH) to concentrate and purify target nucleic acids prior to real‐time PCR. The combination of MCH with multiplex real‐time PCR resulted in a 102–103‐fold increase in detection sensitivity for both pathogens compared to DNA extraction and direct multiplex real‐time PCR. The detection threshold observed for the MCH multiplex real‐time PCR assay was a combination of 105C. michiganensis subsp. michiganensis CFU/ml plus a 10−4‐fold dilution of total RNA extracted from PepMV‐infected tomato leaf tissue. These observations provide proof for the concept that MCH can facilitate the simultaneous detection of Clavibacter michiganensis subsp. michiganensis and PepMV by multiplex real‐time PCR.

A Rapid, Sensitive Assay for Ralstonia solanacearum Race 3 Biovar 2 in Plant and Soil Samples Using Magnetic Beads and Real-Time PCR.

The Ralstonia solanacearum species complex causes economically significant diseases in many plant families worldwide. Although generally limited to the tropics and subtropics, strains designated race 3 biovar 2 (R3Bv2) cause disease in cooler tropical highlands and temperate regions. R3Bv2 has not become established in North America but, due to concerns that it could devastate the U.S. potato industry, it has been designated a Select Agent, and is subject to strict quarantine regulations. Quarantine screening for R3Bv2 requires rapid and robust assays applicable to small populations present in plant tissues or soil, and must distinguish R3Bv2 from the multiple other R. solanacearum subgroups. We developed a 100%-accurate real-time polymerase chain reaction (RT-PCR) assay that can detect R3Bv2 populations >1,000 cells ml-1. However, detection by RT-PCR was inhibited by compounds present in some plant and soil samples. Therefore, we developed simple immunomagnetic separation (IMS) and magnetic capture hybridization (MCH) methods to purify R. solanacearum cells or DNA from PCR inhibitors. When coupled with RT-PCR, these tools permitted detection of R3Bv2 at levels >500 cells ml-1 in stem, tuber, and soil samples when direct RT-PCR failed, and reduced detection time from days to hours. IMS-RT-PCR was usually more sensitive than MCH-RT-PCR, especially at lower population levels.

Magnetic capture hybridization technology: a new tool for selective isolation of cell-free circulating DNA from human plasma

Magnetic capture hybridization technology: a new tool for selective isolation of cell-free circulating DNA from human plasma

Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

PCA3: A urine-based genetic assay for detection of prostate cancer in men with elevated PSA

5054 Background: Currently, early detection of prostate cancer relies primarily on an abnormal digital rectal examination (DRE) and an elevated prostate-specific antigen (PSA) level leading to a prostate biopsy. However, because of low positive predictive values, up to 75% of men with elevated PSA and/or suspicious DRE have a negative biopsy. We investigated the value of a new molecular marker–PCA3 in predicting the likelihood of prostate cancer. Methods: We undertook a prospective, multi-practice, community-urologist-based, and IRB approved clinical trial to evaluate PCA3. Urine samples were obtained from 974 men with elevated serum PSA (&gt; 2.5ng/ml) and/or abnormal digital rectal examination prior to routine minimum 10-core prostate biopsy following standard study protocol in 30 medical practices. Urine samples were processed within 48 hours of collection. PCA3 and PSA mRNA were isolated, amplified and quantified by magnetic target capture, transcription-mediated amplification, and chemiluminescent hybridization protection assay technologies. The PCA3 value was determined using the ratio of PCA3 mRNA copy number to the PSA mRNA copy number multiplied by 1,000. Results: In total, 380 of 974 patients (39%) were diagnosed with prostate cancer, with a mean Gleason score of 7 (range, 6–9) and 26% (range 1–100%) of the biopsy specimens involved by cancer. An additional 106 cases (11%) had only high-grade PIN and/or atypical small acinar proliferation suspicious for cancer (ASAP), and 488 cases (50%) were benign. The mean PCA3 value in men with prostate cancer was significantly higher than in those without cancer (48 vs. 26, p &lt; 0.0001). PCA3 score was associated with the presence of cancer (p &lt; 0.0001) and Gleason score (p = 0.0001), but was not associated cancer volume (p = 0.56). Using a cutoff value of 35, PCA3 had an odds ratio of 2.6 for predicting prostate cancer. PCA3 had a specificity of 77% and a sensitivity of 44% for the diagnosis of prostate cancer, while the specificity and sensitivity for serum PSA were 22% and 87%, respectively. Conclusions: We found that the PCA3 urine test significantly improved the specificity for the detection of prostate cancer compared to serum PSA. Further evaluation of this urinary marker in screening for prostate cancer appears indicated. No significant financial relationships to disclose.

Specific magnetic bead-based capture of free fetal DNA from maternal plasma

An automated magnetic capture hybridization (MCH) method for the extraction and enrichment of fetal RHD specific DNA fragments from maternal plasma was developed using plasma from 1000 D-negative pregnant women. A real time PCR protocol for RHD exon 7 was applied. MCH was compared with the QIAamp DSP Virus Kit (QIAamp) as a reference. Compared with the QIAamp method, the percentage of fetal DNA increased from 2.86% to 4.83% ( p < 0.05, n = 8). The 95% detection limit of MCH was determined at 286 pg/ml (43 geg/ml) compared with 138 pg/ml (21 geq/ml) for the QIAamp DSP Virus Kit.

Detection of Salmonella and simultaneous detection of Salmonella and Shiga-like toxin-producing Escherichia coli using the magnetic capture hybridization polymerase chain reaction

J. CHEN AND M.W. GRIFFITHS. 2001. The magnetic capture hybridization polymerase chain reaction (MCH-PCR) was used to detect Salmonella and also to simultaneously detect Salmonella and Shiga-like toxin-producing Escherichia coli (SLTEC). Fifty-seven Salmonella and 41 SLTEC were included in the study. Salmonella were detected either individually by a single MCH-PCR assay targeting the inv gene or simultaneously with SLTEC by a multiplex MCH-PCR in which SLTEC were detected using primers for the slt genes. Both single and multiplex assays were found to be specific for tested pathogens. The results indicate that MCH-PCR can be used as means of detecting single or multiple bacterial pathogen(s).

A semi-automated and highly sensitive streptavidin magnetic capture-hybridization RT-PCR assay: Application to genus-wide or species-specific detection of several viruses of ornamental bulb crops

A semi-automated, rapid and sensitive method that combines magnetic capture-hybridization and reverse-transcription polymerase chain reaction (MCH/RT-PCR) for the detection of plant viruses is described. The assay uses a target specific biotin-labelled oligoprobe for RNA capture and streptavidin-coated magnetic beads for subsequent RNA-oligoprobe hybrid isolation from plant lysate. Detection and specific identification was accomplished by RT-PCR. This approach was investigated for the specific detection of Tobacco rattle virus and for the detection of viruses within the Potexvirus group in leaves, dormant bulbs and corms of flower bulbs of different species. Dilution series of TRV-infected tulip leaf sap showed that MCH/RT-PCR was 70,588 times more sensitive than enzyme-linked immunosorbent assay (ELISA) and was similar to that of RT-PCR. ELISA underestimated the infection levels of TRV in field samples compared to MCH/RT-PCR. The ability of MCH/RT-PCR to be performed in a microtiter plate on an automatic nucleic acid isolation station facilitates high throughput virus diagnostics. RNA isolation and purification was rapid, specific, sensitive, contamination-free and reproducible making this method amenable for routine indexing of stock plants as part of a management plan to reduce the propagation of virus-infected plants.

Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR

Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 × 10 4 Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 °C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.

Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or real-time PCR

The removal of target DNA by magnetic capture hybridization (MCH) from constituents inhibitory to amplification by polymerase chain reaction (PCR) was evaluated using Salmonella as the test pathogen. Hybrids were subjected to both conventional and quantitative real-time PCR (qPCR). When PCR inhibitors commonly found in water were added to the reaction, MCH-PCR increased the detection sensitivity on the order of 8 to 2,000-fold compared with the system using only PCR. To determine the selectivity of MCH for target DNA (Salmonella), different amounts of non-target DNA (Escherichia coli) were added to the qPCR reaction. The highest non-target DNA concentration interfered with the amplification by qPCR alone, while MCH-qPCR was unaffected. Average recovery of Salmonella DNA by MCH-qPCR was 31% using optimized buffers, washing solutions and enzymatic digestion. A recovery function was proposed in order to calculate the real cell number based on the measured value. Preliminary testing confirmed the suitability of this method for analysis of natural waters.