Standard Polymerase Chain Reaction Assay Research Articles

Neutralizing Antibody Levels as a Correlate of Protection Against SARS-CoV-2 Infection: AModeling Analysis.

Although anti-severe acute respiratory syndrome-coronavirus 2 antibody kinetics have been described in large populations of vaccinated individuals, we still poorly understand how they evolve during a natural infection and how this impacts viral clearance. For that purpose, we analyzed the kinetics of both viral load and neutralizing antibody levels in a prospective cohort of individuals during acute infection with alpha variant. Using a mathematical model, we show that the progressive increase in neutralizing antibodies leads to a shortening of the half-life of both infected cells and infectious viral particles. We estimated that the neutralizing activity reached 90% of its maximal level within 11 days after symptom onset and could reduce the half-life of both infected cells and circulating virus by a 6-fold factor, thus playing a key role to achieve rapid viral clearance. Using this model, we conducted a simulation study to predict in a more general context the protection conferred by pre-existing neutralization titers, due to either vaccination or prior infection. We predicted that a neutralizing activity, as measured by 50% effective dose > 103 , could reduce by 46% the risk of having viral load detectable by standard polymerase chain reaction assays and by 98% the risk of having viral load above the threshold of infectiousness. Our model shows that neutralizing activity could be used to define correlates of protection against infection and transmission.

Distribution and Detectability of EGFR Exon 20 Insertion Variants in NSCLC

IntroductionEGFR exon 20 insertion (ex20ins) mutations represent 5% to 10% of EGFR mutations in NSCLC. Identifying patients with EGFR ex20ins is challenging owing to the limited coverage of polymerase chain reaction (PCR) assays and the relatively recent use of next-generation sequencing (NGS). This study analyzes the spectrum of EGFR ex20ins variants in a large patient population from a global clinical trial and several real-world cohorts and the ability of PCR kits to identify these alterations. MethodsWe conducted this retrospective analysis in patients with NSCLC who underwent NGS or other sequencing testing and had a known EGFR ex20ins mutation. Patients were gathered from a clinical trial (NCT02716116), a chart review study in Germany, and the LC-SCRUM-Japan, GENIE, and U.S. COTA databases. Proportions of patients with ex20ins variants that could have been detected by six commercially available and widely used PCR kits were calculated in each data set. ResultsOverall, 636 patients with NSCLC harboring EGFR ex20ins mutations were included in this analysis and 104 unique EGFR ex20ins variants were identified across the data sources. The proportion of patients whose ex20ins could have been detected by any PCR test alone ranged from 11.8% to 58.9% across the data sources. ConclusionsOur findings suggest that the PCR tests evaluated would have missed more than 40% of patients with NSCLC harboring EGFR ex20ins mutations. NGS-based genetic testing is preferable than standard PCR assays and can substantially improve the identification of the diverse profile of EGFR ex20ins variants in NSCLC.

The Active Surveillance of Staphylococcus aureus using Polymerase Chain Reaction-based Identification Method among Hospitalized-patient of Haji Adam Malik General Hospital, Medan, Indonesia

BACKGROUND: Active surveillance of methicillin-resistant Staphylococcus aureus (MRSA) carriers is associated with the lower incidence of bacteremia and lower mortality rates throughout literature; yet, this important step still remains problematic for developing countries, particularly Indonesia. AIM: The study aimed to demonstrate MRSA colonization rate in Haji Adam Malik Hospital, Medan, Indonesia. MATERIALS AND METHODS: The study enrolled 200 mucocutaneous isolates obtained from hospitalized patients during a 1-year period of study (2018). VITEK-2 system in addition to standard bacterial identification, such as gram staining, latex agglutination test, and hemolysis pattern, was performed to select S. aureus colonies in two different laboratories, Microbiology laboratory of Haji Adam Malik General Hospital and Multidisciplinary Laboratory, Faculty of Medicine, Universitas Sumatera Utara, for polymerase chain reaction (PCR) examination. RESULTS: Based on the VITEK-2 system preliminary identification, there were 80 S. aureus colonies which then underwent PCR examination. Through standard PCR assay, there were 32 bacterial isolates contained the mecA gene and it can be determined MRSA colonization rate of the hospital was 16% with consistent results of standard bacterial identification. CONCLUSIONS: Active surveillance of MRSA carriers is mandatory and urged it as a regular program in a hospital setting to decrease MRSA transmission rate.

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay.

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

Development and Clinical Validation of Iso-IMRS: A Novel Diagnostic Assay for P. falciparum Malaria.

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/μL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection.

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.

A Nuclease Protection ELISA Assay for Colorimetric and Electrochemical Detection of Nucleic Acids.

Early and accurate diagnosis is crucial to monitor infection outcomes and provide timely interventions. However, gold standard polymerase chain reaction assays (PCR) are labor-intensive and require expensive reagents and instrumentation. Nuclease protection has been used for decades to detect and quantify nucleic acid but has not yet been investigated as a diagnostic tool for infectious disease. In this work, we describe a nuclease protection enzyme-linked immunosorbent assay (NP-ELISA) for accurate and sensitive detection of nucleic acid. Briefly, binding of a nucleic acid target to an oligo probe protects it from digestion of un-hybridized nucleic acid by S1 nuclease. Following the workflow of an ELISA, a horseradish peroxidase (HRP)-conjugated antibody binds the probe and oxidizes its substrate to generate signal. The assay was validated with three HRP substrates for absorbance, chemiluminescence, and electrochemical readouts, demonstrating great versatility. Electrochemical detection with 3,3',5,5'-Tetramethylbenzidine (TMB) gave the highest assay sensitivity with a limit of detection of 3.72×103 molecules mL-1. Furthermore, non-complementary targets did not generate a response, indicating a high degree of specificity. This proof of principle serves as a stepping stone towards developing miniaturized, multiplexed nuclease protection assays for point-of-care diagnosis.

The importance of morphological identification of African anopheline mosquitoes (Diptera: Culicidae) for malaria control programmes

BackgroundThe correct identification of disease vectors is the first step towards implementing an effective control programme. Traditionally, for malaria control, this was based on the morphological differences observed in the adults and larvae between different mosquito species. However, the discovery of species complexes meant that genetic tools were needed to separate the sibling species and today there are standard molecular techniques that are used to identify the two major malaria vector groups of mosquitoes. On the assumption that species-diagnostic DNA polymerase chain reaction (PCR) assays are highly species-specific, experiments were conducted to investigate what would happen if non-vector species were randomly included in the molecular assays.MethodsMorphological keys for the Afrotropical Anophelinae were used to provide the a priori identifications. All mosquito specimens were then subjected to the standard PCR assays for members of the Anopheles gambiae complex and Anopheles funestus group.ResultsOne hundred and fifty mosquitoes belonging to 11 morphological species were processed. Three species (Anopheles pretoriensis, Anopheles rufipes and Anopheles rhodesiensis) amplified members of the An. funestus group and four species (An. pretoriensis, An. rufipes, Anopheles listeri and Anopheles squamosus) amplified members of the An. gambiae complex.ConclusionsMorphological identification of mosquitoes prior to PCR assays not only saves time and money in the laboratory, but also ensures that data received by malaria vector control programmes are useful for targeting the major vectors.

Performance of Loop-Mediated Isothermal Amplification for the Identification of Submicroscopic Plasmodium falciparum Infection in Uganda.

Accurately identifying and targeting the human reservoir of malaria parasitemia is critical for malaria control, and requires a reliable and sensitive diagnostic method. Loop-mediated isothermal amplification (LAMP) is increasingly used to diagnose submicroscopic parasitemia. Although most published studies report the sensitivity of LAMP compared with nested polymerase chain reaction (PCR) as ≥ 80%, they have failed to use a consistent, sensitive diagnostic as a comparator. We used cross-sectional samples from children and adults in Tororo, Uganda, a region with high but declining transmission due to indoor residual spraying, to characterize the sensitivity and specificity of pan-Plasmodium LAMP for detecting submicroscopic infections. We compared LAMP results targeting a mitochondrial DNA sequence conserved in all Plasmodium species, performed on DNA extracted from dried blood spots, to those of a gold standard quantitative PCR assay targeting the var gene acidic terminal sequence of Plasmodium falciparum (varATS qPCR), performed on DNA extracted from 200 µL of whole blood. Using LAMP and varATS qPCR increased the detection of parasitemia 2- to 5-fold, compared with microscopy. Among microscopy-negative samples, the sensitivity of LAMP was 81.5% for detecting infection ≥ 1 parasites/µL. However, low density infections were common, and LAMP failed to identify more than half of all infections diagnosed by varATS qPCR, performing with an overall sensitivity of 44.7% for detecting submicroscopic infections ≥ 0.01 parasites/µL. Thus, although the LAMP assay is more sensitive than microscopy, it missed a significant portion of the submicroscopic reservoir. These findings have important implications for malaria control, particularly in settings where low-density infections predominate.

Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

Real-time PCR assay to detect Verticillium albo-atrum and V. dahliae in hops: development and comparison with a standard PCR method

Verticillium wilt, caused by Verticillium albo-atrum and V. dahliae, is a devastating disease that causes considerable economic crop losses in hop fields. The fungus can survive in soil for several years by producing resting structures, and due to the lack of effective fungicides, the disease is spreading. Thus, a fast and sensitive detection system is urgently needed. In this study we report a novel routine detection method for the identification of V. albo-atrum and V. dahliae on a molecular basis. The standard polymerase chain reaction (PCR) assay includes isolation of the fungus from affected tissue by following DNA extraction from fungal cultures and PCR identification by specific primers. In order to improve this detection assay, DNA was isolated directly from the hop bine using a commercially available DNA isolation kit. A multiplex real-time PCR assay for the simultaneous detection of V. albo-atrum and V. dahliae was established using a specific primer pairs/TaqMan probes combination. Ninety-six plants were collected from different culti-vars and locations in Germany and tested by standard and real-time PCR assay. The new detection system is more accurate, sensitive and time-saving than the standard PCR method and is suitable for routine use. The test provides a valuable tool for rapid and sensitive detection of V. albo-atrum and V. dahliae in plants and gives farmers and plant protection offices the chance to react accordingly and to evaluate measures for plant disease management against Verticillium.

Human Papillomavirus in Older Women: New Infection or Reactivation?

Human Papillomavirus in Older Women: New Infection or Reactivation?

Mosquito bisection as a variable in estimates of PCR-derived malaria sporozoite rates

BackgroundHighly sensitive polymerase chain reaction (PCR) methods offer an alternative to the light microscopy examination of mosquito salivary glands for the determination of malaria sporozoite rates in wild caught female Anopheles. Removal of mosquito abdomens is assumed to eliminate false positives caused by malaria oocyst DNA in the midgut. This assumption has not been tested with current gold standard PCR assays, and for the variety of conditions that specimens could encounter in the laboratory and field.MethodsLaboratory Anopheles stephensi were used that had been infected with Plasmodium falciparum 6–7 days and 14 days post infection (p.i.), when oocysts only and oocysts + sporozoites, respectively, are developed. Mosquitoes were killed and immediately frozen, air dried before being frozen, or stored under humid conditions overnight before being frozen, to simulate a range of conditions in the field. Additionally, abdomens were removed anterior to, at, or posterior to the junction of the abdomen and thorax, and both portions were processed using a standard nested PCR of the small sub-unit nuclear ribosomal genes (ssrDNA) with products visualized on agarose gels.ResultsOverall, 4.1 % (4/97) of head + thorax samples that were 6–7 days p.i. gave apparent false positives for sporozoites, compared to 9.3 % (9/97) that were positive for abdomens. No positives (0/52) were obtained when similar specimens were bisected anterior to the junction of the thorax and abdomen, compared to 21.2 % (11/52) that were positive for posterior portions. Multiple bands were noted for positives from the ‘Frozen’ treatment and the rate of false negatives due to DNA degradation appears higher under the ‘Humid’ treatment. Reproducibility of results for the ‘Frozen’ treatment was 90 %.ConclusionsDespite the importance of specimen condition and the bisection step in determining sporozoite rates, little attention has been paid to them in the literature. Recommendations from this study are that: 1) care needs to be taken to reduce DNA degradation in the field; 2) mosquito abdomens be separated anterior to the junction of the thorax and abdomen; and 3) DNA sequencing of a subsample of positive results should be undertaken if possible.

Development of a set of multiplex standard polymerase chain reaction assays for the identification of infectious agents from aborted bovine clinical samples

The current study describes the development of a set of 5 multiplex polymerase chain reaction (mPCR) assays for the simultaneous detection of abortive infection agents in bovine fetal tissues, including Brucella spp., Leptospira spp., and Campylobacter fetus (mPCR1); Hammondia heydorni, Neospora caninum, and Toxoplasma gondii (mPCR2); Coxiella burnetii and Chlamydophila psittaci (mPCR3); Mycoplasma bovis, Mycoplasma bovigenitalium, and Ureaplasma diversum (mPCR4); and Bovine viral diarrhea virus (BVDV) and Bovine herpesvirus-1 (BoHV-1; mPCR5). The protocol was tested on different tissue samples collected from 50 aborted bovine fetuses, and it showed that out of the 50 fetuses, 7 (14%, mPCR2) were PCR-positive for N. caninum, 4 (8%, mPCR5) were PCR-positive for BVDV, and 2 (4%, mPCR4) were PCR-positive for U. diversum. The results obtained by using each multiplex PCR were 100% concordant with those obtained by using the respective PCR assays targeting single genes on the same specimens. Moreover, all multiplex PCR assays on clinical samples were compared with reference methods, obtaining a perfect accordance in all samples and confirming the validity of the set of multiplex PCR assays. The proposed set of multiplex PCR assays is, therefore, suitable for the simultaneous detection of the main infectious agents responsible for bovine abortion.

PCR detection of Streptococcus mutans and Aggregatibacter actinomycetemcomitans in dental plaque samples from Haitian adolescents

Streptococcus mutans and Aggregatibacter actinomycetemcomitans are oral pathogens associated with dental caries and periodontitis, respectively. The aim of this study was to determine the colonization of these two microorganisms in the dental plaque of a group of Haitian adolescents using two different polymerase chain reaction (PCR) methods, standard PCR, and quantitative real-time PCR (qPCR) assays. Fifty-four pooled supra-gingival plaque samples and 98 pooled sub-gingival plaque samples were obtained from 104 12- to19-year-old rural-dwelling Haitians. The total genomic DNA of bacteria was isolated from these samples, and all participants also received caries and periodontal examinations. Caries prevalence was 42.2%, and the mean decayed, missing, and filled surface (DMFS) was 2.67 ± 5.3. More than half of the adolescents (53.3%) experienced periodontal pockets (Community Periodontal Index score ≥3). S. mutans was detected in 67.3% by qPCR and 38.8% by PCR of the supra-gingival plaque samples (p < 0.01), and 36.6% by qPCR and 8.1% by PCR of the sub-gingival samples (p < 0.01). A. actinomycetemcomitans was detected in 85.1% by qPCR and 44.0% by PCR of the sub-gingival samples (p < 0.01), but the prevalence was similar, 67.3% by qPCR and 59.2% by PCR, in the supra-gingival plaque samples. Neither age nor gender was significantly correlated to the bacterial colonization. The results demonstrated a moderate-to-high prevalence of S. mutans and A. actinomycetemcomitans in the Haitian adolescent population, and qPCR is more sensitive than standard PCR in field conditions. These findings suggest that qPCR should be considered for field oral epidemiologic studies and may be necessary in investigations having major logistic challenges.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Detection and Characterization of cry1Ac Transgene Construct in Bt Cotton: Multiple Polymerase Chain Reaction Approach

To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.

Multi-Node Graphs: A Framework for Multiplexed Biological Assays

Multiplex polymerase chain reaction (PCR) is an extension of the standard PCR protocol in which primers for multiple DNA loci are pooled together within a single reaction tube, enabling simultaneous sequence amplification, thus reducing costs and saving time. Potential cost saving and throughput improvements directly depend on the level of multiplexing achieved. Designing reliable and highly multiplexed assays is challenging because primers that are pooled together in a single reaction tube may cross-hybridize, though this can be addressed either by modifying the choice of primers for one or more amplicons, or by altering the way in which DNA loci are partitioned into separate reaction tubes. In this paper, we introduce a new graph formalism called a multi-node graph, and describe its application to the analysis of multiplex PCR scalability. We show, using random multi-node graphs that the scalability of multiplex PCR is constrained by a phase transition, suggesting fundamental limits on efforts to improve the cost-effectiveness and throughput of standard multiplex PCR assays. In particular, we show that when the multiplexing level of the reaction tubes is roughly theta(log (sn)) (where s is the number of primer pair candidates per locus and n is the number of loci to be amplified), then with very high probability we can 'cover' all loci with a valid assignment to one of the tubes in the assay. However, when the multiplexing level of the tube exceeds these bounds, there is no possible cover and moreover the size of the cover drops dramatically. Simulations using a simple greedy algorithm on real DNA data also confirm the presence of this phase transition. Our theoretical results suggest, however, that the resulting phase transition is a fundamental characteristic of the problem, implying intrinsic limits on the development of future assay design algorithms.

ThirdBorreliaSpecies in White-footed Mice

Third<i>Borrelia</i>Species in White-footed Mice

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 10 2-fold higher than the standard real-time PCR assay and 10 7-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.