Amplification Control Research Articles

One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5′-end and 3′-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/μl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

Генотипування лістерій за допомогою полімеразної ланцюгової реакції (ПЛР) та його епідеміологічне значення

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis

Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens.

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

Nucleic Acid-Based Lateral Flow Biosensor for Salmonella Typhi and Salmonella Paratyphi: A Detection in Stool Samples of Suspected Carriers.

A multiplex rapid detection system, based on a PCR-lateral flow biosensor (mPCR-LFB) was developed to identify Salmonella Typhi and Salmonella Paratyphi A from suspected carriers. The lower detection limit for S. Typhi and S. Paratyphi A was 0.16 and 0.08 ng DNA equivalent to 10 and 102 CFU/mL, respectively. Lateral flow biosensor was used for visual detection of mPCR amplicons (stgA, SPAint, ompC, internal amplification control) by labeling forward primers with fluorescein-isothiocyanate (FITC), Texas Red, dinitrophenol (DNP) and digoxigenin (DIG) and reverse primers with biotin. Binding of streptavidin-colloidal gold conjugate with the amplicons resulted in formation of a red color dots on the strip after 15–20 min of sample exposure. The nucleic acid lateral flow analysis of the mPCR-LFB was better in sensitivity and more rapid than the conventional agarose gel electrophoresis. Moreover, the mPCR-LFB showed 100% sensitivity and specificity when evaluated with stools spiked with 100 isolates of Salmonella genus and other bacteria. A prospective cohort study on stool samples of 1176 food handlers in outbreak areas (suspected carriers) resulted in 23 (2%) positive for S. Typhi. The developed assay has potential to be used for rapid detection of typhoid carriers in surveillance program.

Theoretical study of the control of absorption, transparency, and amplification in a microwave- and RF-driven four-level (Δ + ∇)-type closed-contour interaction system

This paper describes a theoretical study of the optical responses of an atomic medium to the probe field in a four-level (Δ + ∇)-type closed-contour interaction system driven by two pump laser fields (P1, P2), one probe laser field (Pr) and two microwave or radio frequency (RF) coupling fields (L, U). Here, L and U are considered to connect the forbidden electric dipole transitions, i.e. two hyperfine ground states of the Δ subsystem and two hyperfine excited states of the ∇ subsystem, respectively. Since each subsystem is a closed-loop system, relative phases are thought to be present between the optical fields (pump and probe fields), and the microwave or RF fields. The manipulation of the quantum coherence of the system by the strengths of coupling fields along with the relative phases is demonstrated in terms of the changes occurring in the absorption of the probe field by the medium. The probe absorption and dispersion profiles are extracted by numerically solving the optical Bloch equations for the system under steady-state conditions. In this study, we start by illuminating the system with the probe only, and then, by applying the pump fields and the coupling fields one by one, the changes in the absorptive and dispersive probe line profiles are investigated for two distinct cases that depend on the relative strengths of the pump fields, e.g. when P2 is stronger than P1 and vice versa. Furthermore, the individual effects of the strengths and phases of the coupling fields on the probe absorption, transparency and amplification are also explored for both cases. All the changes that appear in the probe signal as a result of the application of different fields are explained by the modified interaction fields or the corresponding effective Rabi frequencies obtained from the partial dressed-state analysis.

Development of a disposable cartridge real-time PCR test for detection of Chlamydia trachomatis directly from urine

Abstract We present a PCR assay screening Chlamydia trachomatis directly from urine without pre-amplification or sample clean-up and demonstrate suitability for implementation on a disposable cartridge system. Primers and probes were designed to detect all Chlamydia trachomatis serovars with a high degree of sensitivity and specificity. The assay also incorporated a synthetic internal amplification control to monitor inhibition of the PCR reaction. The assay protocol worked directly on urine samples with initial heating to 85 °C for ten minutes, followed by a duplex realtime PCR (95 °C, 55 °C, 45 cycles) on resulting urine lysate. The test cartridge incorporated heating and liquid manipulation using pinch valves and a compressible blister pouch. Lyophilised PCR reagents were incorporated into the cartridge and were re-suspended by a 40 μL aliquot of the urine lysate. Primers were designed to screen for all Chlamydia trachomatis serovars with a high degree of sensitivity and specificity. Evaluation of the newly developed PCR assay was carried out on the LightCycler™ using urine samples previously confirmed positive or negative for the target pathogen, using the ABBOTT real-time PCR Cobas CT/NG test at a local hospital.

Standardized Shiga-Toxin Encoding Genes Real-Time PCR Screening Methods Comparison and Development of an Internally Controlled Assay for Pan-stx2 Detection.

Various primer and probe sets have been developed and standardized, but certain sets may have low efficiency or miss some stx-subtypes. To compare the efficiency of the recommended stx screening primers and probe sets in four standardized methods and develop a new primers and probe system with an internal amplification control (IAC) for all known stx2 subtypes. The inclusivity and specificity of recommended screening primers and probe sets in four standardized methods were compared. A new pan-stx2 primer and probe set was adapted from the International Organization for Standardization (ISO) method for all known stx2 subtypes. The robustness of the new method was assessed in seven laboratories and also assessed in ground beef and bean sprout samples. None of the recommended screening primers and probe sets in the four standardized methods could efficiently amplify all the stx2 subtypes because of various mismatches in the primers or the probe sequences. A new primers and probe system adapted from the ISO method, through introducing degenerate bases in primers and probe sequences with an IAC, showed high amplification efficiency and specificity for all known stx2 subtypes in ground beef and bean sprouts samples. The specificity of the new method was assessed in seven laboratories and showed robust and consistent results. This study provided evidence for Shiga-toxin producing Escherichia coli (STEC) screening method development, and the newly developed primers and probes system should be considered in the revision of the standardized methods. None of the recommended screening primer and probe set in the four official methods could efficiently amplify all the stx2 subtypes. A new developed primer and probe set showed high amplification efficiency and specificity for all known stx2 subtypes in fresh ground beef and bean sprouts samples. The newly developed stx2 screening system showed robustness and consistency during interlaboratory study.

Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay.

The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

One-step differential detection of Salmonella enterica serovar Typhi, serovar Paratyphi A and other Salmonella spp. by using a quadruplex real-time PCR assay

Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 103 –104 CFU/mL (or g) and a high PCR efficiency with different targets (R2 > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.

Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR

In this study, the detection of PRV DNA in nasal swab (n = 440) and oral fluid (n = 1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs (n = 40) was comparatively evaluated by real-time PCR. Serum samples (n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were re-expressed as “efficiency standardized Cqs (ECqs)” as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85 % vs 83 %) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100 % and diagnostic sensitivities for oral fluid and nasal swab specimens of 53 % (95 % CI: 43 %, 62 %) and 70 % (95 % CI: 55 %, 83 %), respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.

Loop-mediated isothermal amplification combined with immunomagnetic separation and propidium monoazide for the specific detection of viable Listeria monocytogenes in milk products, with an internal amplification control

Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.

Surface Plasmon-Photon Coupling in Lanthanide-Doped Nanoparticles.

Lanthanide-doped nanoparticles have great potential for energy conversion applications, as their optical properties can be precisely controlled by varying the doping composition, concentration, and surface structures, as well as through plasmonic coupling. In this Perspective we highlight recent advances in upconversion emission modulation enabled by coupling upconversion nanoparticles with well-defined plasmonic nanostructures. We emphasize fundamental understanding of luminescence enhancement, monochromatic emission amplification, lifetime tuning, and polarization control at nanoscale. The interplay between localized surface plasmons and absorbed photons at the plasmonic metal-lanthanide interface substantially enriches the interpretation of plasmon-coupled nonlinear photophysical processes. These studies will enable novel functional nanomaterials or nanostructures to be designed for a multitude of technological applications, including biomedicine, lasing, optogenetics, super-resolution imaging, photovoltaics, and photocatalysis.

Target-Cell-Specific Bioorthogonal and Endogenous ATP Control of Signal Amplification for Intracellular MicroRNA Imaging

A stringent signal amplification method to profile microRNA (miRNA) expression within a specific cell remains a key challenge in biology. To address this issue, we report a target-cell-specific DNA nanosystem for endogenous adenosine-5'-triphosphate (ATP) bioorthogonal activation of the hybridization chain reaction (HCR) to spatiotemporally controlled signal amplification detection of miRNA in vitro and in vivo. The system consists of ATP aptamer-sealed engineered HCR functional units combined with a cancer cell membrane-encapsulated glutathione (GSH)-responsive metal-organic framework (MOF). Once the nanosystem is specifically and efficiently internalized into a cancer cell through membrane-mediated homing targeting, the MOF structure degrades and releases HCR functional units. The endogenous high expressional ATP recognizes the aptamer, allowing the HCR functional units to adopt its active modality. The activated HCR functional units are then able to spatiotemporally and bioorthogonally image miRNA with high sensitivity in vitro and in vivo.

The presence and genetic characteristics of porcine circovirus 3 from pigs in Southern and Central provinces of Vietnam

Porcine circovirus type 3 (PCV3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. This study was carried out in order to investigate the presence and further genetic characteristics of PCV3 from swine herds in Southern and Central provinces of Vietnam. A duplex PCR assay for rapid detection of PCV3 in pigs was established with a pair of specific primers designed between rep and cap gene segment to amplify full-length ORF2 and another set of primers binding to COX1gene serving as an internal amplification control (IAC). The resulting duplex PCR was used to examine PCV3 presence in 94tissue and serum samples. Subsequently, PCV3 was detected in 10 out of 94 cases (10.6%). The infection rate in sows (14.3%) was higher than that in grower pigs (7.7%). Regarding nucleotide sequence comparison, 10 ORF2 genes were selected for nucleotide sequencing and their alignment showed 97.2% - 99.5% homology. According to the phylogenetic analysis and sequence alignment of cap gene, all the sequences were clustered into group PCV3a,including 9 strains of sub-group PCV3a1 and only one strain of subgroup PCV3a2. These findings indicated that the PCV3a group is circulating in swine farms in Vietnam. This study provides better insights into epidemiology of this pathogen in the national swine industry.

Controlling Ultra-Large Optical Asymmetry in Amorphous Molecular Aggregations.

Although ultra-large optical asymmetry appears in crystalline materials, distractions from the mesoscopic ordering often causes inauthenticity in chiropticity. In amorphous materials, however, it remains challenging and elusive to achieve large chiropticity. Herein, we report the quantitative control of chiral amplification, on amorphous supramolecular structures of cholesteryl-linked bis(dipyrrinato)zinc(II), to an exceptionally high level. A proper chiral packing of the building block at several molecular scale considerably contributes to the absorptive dissymmetry factor gabs , although the system is overall disordered. The intense and tunable aggregation strength renders a variable gabs value up to +0.10 and +0.31 in the solution and in film state. On this basis, a superior ON-OFF switching of chiropticity is realized under external stimuli. This work establishes a general design principle to control over ultra-large optical asymmetry on a wider scope of chiral materials.

Controlling Ultra‐Large Optical Asymmetry in Amorphous Molecular Aggregations

AbstractAlthough ultra‐large optical asymmetry appears in crystalline materials, distractions from the mesoscopic ordering often causes inauthenticity in chiropticity. In amorphous materials, however, it remains challenging and elusive to achieve large chiropticity. Herein, we report the quantitative control of chiral amplification, on amorphous supramolecular structures of cholesteryl‐linked bis(dipyrrinato)zinc(II), to an exceptionally high level. A proper chiral packing of the building block at several molecular scale considerably contributes to the absorptive dissymmetry factor gabs, although the system is overall disordered. The intense and tunable aggregation strength renders a variable gabs value up to +0.10 and +0.31 in the solution and in film state. On this basis, a superior ON‐OFF switching of chiropticity is realized under external stimuli. This work establishes a general design principle to control over ultra‐large optical asymmetry on a wider scope of chiral materials.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Electric steering of spin excitation in nanostructured synthetic antiferromagnet

Two or more ferromagnetic layers separated by metallic nanoscale spacers may couple antiferromagnetically forming a synthetic antiferromagnet (SAF) with versatile functionalities that are derived from their ferro- and antiferromagnetic nature. Here, we present a nanostructure consisting of a SAF deposited on a conductive substrate with strong spin–orbit coupling. As demonstrated analytically and fully numerically, the system exhibits exceptional points at which a charge current flowing in the substrate results in extraordinary characteristics of magnetic excitation and signal propagation in the SAF. We predict local electric steering, amplification, and damping of magnonic excitations by tuning the voltage on the substrate. Electrical amplification and local control of spin-pumping currents are also demonstrated. The results point to an alternative way for electrical spatiotemporal control of the nanoscale SAF with promising applications in spintronics and spin-based logic computing.

A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne's Disease.

Johne's disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration-validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method.