Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation.

BACKGROUND: Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. RESULTS: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55-80%. CONCLUSIONS: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications.

BACKGROUND: One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing. METHODS AND RESULTS: We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing. CONCLUSIONS: The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

BACKGROUND: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. METHODS AND RESULTS: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. CONCLUSIONS: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

Development of a Simple Direct and Hot-Start PCR Using Escherichia coli-Expressing Taq DNA Polymerase.

Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli (E. coli) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli-expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.

Polydopamine nanospheres-assisted direct PCR for rapid detection of Escherichia coli O157:H7.

Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 µg/µL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 104 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 105 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 104-5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90 °C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·µL-1 (4 copies·µL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

A Novel PCR Method for Detecting ACE Gene Insertion/Deletion Polymorphisms and its Clinical Application.

BACKGROUND: Angiotensin-converting enzyme (ACE) plays a major role in blood pressure regulation and cardiovascular homeostasis. The wide distribution and multifunctional properties of ACE suggest it's involvement in various pathophysiological conditions. RESULTS: In this study, a novel visual detection method for ACE I/D polymorphisms was designed by integrating direct PCR without the need for DNA extraction using gold magnetic nanoparticles (GMNPs)-based lateral flow assay (LFA) biosensor. The entire detection procedure could enable the genotyping of clinical samples in about 80 min. The detection limit was 0.75 ng and results could be obtained in 5 min using the LFA device. Three hundred peripheral blood samples were analyzed using the direct PCR-LFA system and then verified by sequencing to determine accuracy and repeatability. A clinical preliminary study was then performed to analyze a total of 633 clinical samples. CONCLUSIONS: After grouping based on age, we found a significant difference between the genotypes and the age of patients in the CHD group. The introduction of this method into clinical practice may be helpful for the diagnosis of diseases caused by large fragment gene insertions/deletions.

Direct PCR on Tissue Samples To Detect Mycobacterium tuberculosis Complex: an Alternative to the Bacteriological Culture.

Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union. Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparative to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an interlaboratory trial between European Union National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic equivalents. Diagnostic sensitivity (Se) and specificity (Sp) were, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as Mycobacterium marinum and "Mycobacterium avium subsp. hominissuis," and whole-genome sequencing (WGS) analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture, and the strategy of use should be defined depending on the control or eradication programs.

The development of miniSTRs as a method for high-speed direct PCR.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods.

Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology.

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics.

Direct PCR assays without DNA extraction for rapid detection of hemoglobin Constant Spring and Pakse' genes: application for carrier screening and prenatal diagnosis.

Hemoglobin Constant Spring (Hb CS) and Hb Pakse' (PS) are the common non-deletional α+-thalassemia found in Thailand. These two variants can cause severe thalassemia syndromes, especially in fetus and neonate. Molecular diagnosis is the only confirmatory method because Hb CS and Hb PS are usually missed by routine screening and Hb analysis. Therefore, we aimed to develop rapid direct PCR for the diagnosis of Hb CS and PS genes. Multiplex direct PCR assays for identifying the Hb CS and PS genes in whole blood (WB) and amniotic fluid (AF) specimens were developed. The assays were firstly validated on 290 unrelated whole blood specimens. Hb CS and PS carriers were identified in 67 (23.1%) and 6 (2.1%) cases, respectively. A 100% concordant result as compared to routine PCR assay was observed. The direct PCR assays have been applied successfully for prenatal diagnosis in two families. The result showed that the fetuses were affected by homozygous Hb CS and compound heterozygous Hb CS/Hb PS. Accurate prenatal diagnosis of these families was observed using the newly developed assays. These assays should be applicable in routine thalassemia diagnostics as well as in the large-scale screening of Hb CS and PS in the region.

Two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells in 96-well plates.

BACKGROUND: Although DNA of high quality can be easily prepared from cultured cells with commercially available kits, many studies involve a large number of samples which increases the cost drastically. We optimized two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells directly from wells of 96-well plates. METHODS: Cells (number: 103 -104 ) were lysed with a Direct PCR® lysis buffer or a 10% Chelex100® solution. The lysates were further purified and concentrated by means of DNA precipitation with a blue-colored glycogen as a carrier. PCR and digital PCR were used to evaluate the efficiency of the two methods. RESULTS: For 1000 cells from one primary culture and two tumor cell lines, DNA was reproducible and obtained with recovery rate (obtained/expected amount of DNA) in the range of 50%-90% as measured by the fluorometer dyes instrument Qubit. Using 8 out of a total of 10 µL DNA solution for 1000 cells, both conventional PCR and digital PCR were successful. For digital PCR, more than 1600 positive droplets were obtained for DNA from 1000 cells using the Direct PCR® method, corresponding to a yield efficiency of approximately 80%. Further reducing the number of cells down to 100 would be possible with 160 positive droplets expected. Both reagents are inexpensive (0.08€/sample). CONCLUSIONS: Two methods are efficient, especially the Direct PCR® reagent-based method provides a simple and inexpensive method for preparing DNA suitable for digital PCR from small number of cells.

Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards.

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Copan microFLOQ® Direct Swab collection of bloodstains, saliva, and semen on cotton cloth.

The microFLOQ® Direct Swab was tested by sampling diluted blood, semen, and saliva stains deposited on cotton cloth. DNA typing was performed using the PowerPlex® Fusion 6C System by direct PCR or a modified direct PCR. Direct PCR of swabs sampled the center of a stain, compared to their respective edge samplings, and had higher profile completeness and total relative fluorescent units (RFU) for all dilutions of blood and semen stains tested. The modified direct PCR used template DNA eluted from the swab head using the Casework Direct Kit, Custom and washes either contained 1-thioglycerol (TG) additive or no TG. Modified direct PCR had mixed results for blood, saliva, and semen stains, with semen stains showing significant differences in profile completeness (5% and 1%) and total RFU (neat, 5% and 1%) with the addition of TG to the Casework Direct Reagent. No significant difference was seen in any dilution of blood or saliva stains processed with the modified direct PCR, but profile completeness and total RFU were improved overall compared to stains swabbed with cotton swabs or 4N6FLOQSwabs™. This study supports the hypothesis that the microFLOQ® Direct Swab is able to collect minute amounts of DNA from cotton cloth and may be considered as an alternate pre-screening methodology in forensic biology casework.

An evaluation of direct PCR assays for the detection and quantification of Porphyromonas gingivalis.

Porphyromonas gingivalis has been linked to the development and progression of oesophageal squamous cell carcinoma (ESCC), and is considered to be a high-risk factor for ESCC. Currently, the commonly used methods for P. gingivalis detection are culture or DNA extraction-based, which are either time and labour intensive especially for high-throughput applications. We aimed to establish and evaluate a rapid and sensitive direct quantitative polymerase chain reaction (qPCR) protocol for the detection of P. gingivalis without DNA extraction which is suitable for large-scale epidemiological studies. Paired gingival swab samples from 192 subjects undergoing general medical examinations were analysed using two direct and one extraction-based qPCR assays for P. gingivalis. Tris-EDTA buffer-based direct qPCR (TE-direct qPCR), lysis-based direct qPCR (lysis-direct qPCR) and DNA extraction-based qPCR (kit-qPCR) were used, respectively, in 192, 132 and 60 of these samples for quantification of P. gingivalis. The sensitivity and specificity of TE-direct qPCR was 95.24% and 100% compared with lysis-direct qPCR, which was 100% and 97.30% when compared with kit-qPCR; TE-direct qPCR had an almost perfect agreement with lysis-direct qPCR (κ = 0.954) and kit-qPCR (κ = 0.965). Moreover, the assay time used for TE-direct qPCR was 1.5 h. In conclusion, the TE-direct qPCR assay is a simple and efficient method for the quantification of oral P. gingivalis and showed high sensitivity and specificity compared with routine qPCR.

"The acid test"-validation of the ParaDNA® Body Fluid ID Test for routine forensic casework.

The identification of the cellular origin and composition of crime scene-related traces can provide crucial insight into a crime scene reconstruction. In the last decade, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined. Besides capillary electrophoretic (CE) and real-time quantitative PCR (RT-qPCR)-based approaches for mRNA detection, melt curve analysis bears potential as a simple-to-use method for BFI. The ParaDNA® Body Fluid ID Test relies on HyBeacon® probes and was developed as a rapid test for mRNA-based BFI of six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual, and peripheral blood. The herein presented work was performed as an "acid test" of the system and should clarify whether the approach matches the requirements of forensic routine casework in German police departments. Tested samples consisted of single source as well as of mixed samples.

Prenatal diagnosis of genetic diseases directly using paper-dried cord blood as the starting material for PCR.

A rapid and low-cost method of diagnosis is becoming important for detecting fetal inherited diseases, including single-gene disorders and chromosomal abnormalities. Here, we demonstrated an innovation that use paper-dried cord blood (PCB) as the starting material for PCR and whole genome amplification without any DNA extraction step at a very low cost. A novel PCR buffer named "DDB buffer" containing ammonium sulfate and glycerol were used instead of the conventional 10× PCR buffer. The amplicons were directly analyzed through microchip electrophoresis and whole genome sequencing. Inhibitory substances in filter paper were effectively inactivated using DDB buffer. Direct PCR amplification of DNA fragments ranging from 100 to 900 bp using filter paper spotted with 0.5 to 5 µL of cord blood and various anticoagulants was successful. We were able to determine fetal single-gene disorders and chromosomal diseases in all 46 chromosomes using PCB samples successfully. Compared with prenatal diagnosis using purified DNA, the proposed method is simple, fast, less prone to cross-contamination at minimal cost. Researchers and clinical and healthcare workers may employ this method for genetic diagnosis using cord blood samples with minimum laboratory resources. This method is very promising for a variety of genetic diagnosis applications in underserved communities at the point of need in developing areas. Graphical abstract.

Diagnostic accuracy of two DNA-based molecular assays for detection of porcine circovirus 3 in swine population using Bayesian latent class analysis.

Molecular-based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described 'gold standard' methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV-3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real-time qPCR (qPCR) for detection of PCV-3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84-100) and qPCR (96%; PCI% 90-100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV-3 in Italian swine population. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous discovery of new pathogens poses a challenge in the development and evaluation of adequate diagnostic tools. In fact, since molecular-based tools sometimes are the only available laboratory techniques, it is typically difficult to evaluate their diagnostic performances in the absence of a gold standard. The present study assesses this issue, demonstrating the excellent performances of two PCR-based assays for porcine circovirus 3 (PCV-3) detection using a Bayesian latent class analysis approach. Therefore, the molecular tests evaluated under this study constitute reliable tools for the routine diagnosis and surveillance programs of PCV-3 circulating in swine populations.

Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum.

Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid-cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya , Fyb , Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false-negative rate. With fast turnaround times, the rapid-cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting.