Standard PCR Research Articles

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Establishment and application of a PCR assay for the identification of virulent and attenuated duck plague virus DNA in cotton swabs

Duck plague is an acute, febrile, and septic infectious disease caused by duck plague virus (DPV), which causes serious harm to the duck industry in China. Ducks latently infected with DPV display a clinically healthy state, which is one of the epidemiological characteristics of duck plague. In the present study, to rapidly distinguish vaccine-immunized ducks from wild virus-infected ducks during production, a PCR assay based on the newly identified LORF5 fragment was developed to effectively and accurately identify viral DNA in cotton swab samples and was used to assess artificial infection models and clinical samples. The results showed that the established PCR method had good specificity and that only the virulent and attenuated DNA of duck plague virus was specifically amplified, as the results for the detection of common duck pathogens (duck hepatitis B virus, duck Tembusu virus, duck hepatitis A virus type 1, novel duck reovirus, Riemerella anatipestifer, Pasteurella multocida, and Salmonella) were negative. The amplified fragments of virulent and attenuated strains were 2,454 bp and 525 bp, and their minimum detection amounts were 0.46 pg and 46 pg, respectively. The detection rate of the virulent and attenuated DPV strains in duck oral and cloacal swabs was lower than that of the gold standard PCR method (GB-PCR, which is unable to distinguish virulent and attenuated strains), and cloacal swabs from clinically healthy ducks were more suitable for detection than oral swabs. In conclusion, the PCR assay established in the present study can be used as a simple and effective method for the clinical screening of ducks that are latently infected with virulent strains of DPV and shedding virus, which can provide technical support for the elimination of duck plague from duck farms.

Com probe implemented STexS II greatly enhances specificity in SARS-CoV-2 variant detection

The initial introduction of utilizing double helix structural oligonucleotides known as SNP typing with excellent specificity (STexS) in a standard PCR greatly improved the detection of single nucleotide polymorphisms (SNP) by enhancing amplification rates of primer-matching strands and interrupting mismatched strands by constant instability of kinetics regarding alignment attaching and detaching. The model was beneficial overall in detecting SNP variants consisting of large amounts of wildtype strands such as EGFR mutation genotyping for early detection of non-small cell lung cancer. While the STexS PCR is advantageous in detecting SNPs and biomarkers, limitations were yet observed. Despite the ability to detect variants 10 times more effective than a typical amplification-refractory mutation system PCR, it could only perform optimally in DNA concentrations around 101 ~ 105. To further enhance STexS specificity to perform detecting viral-RNA variants such as the infamous SARS-CoV-2, a novel improvement of the regular TaqMan Probe using Com-probes to inhibit high copy wild targets and amplify low copy mutant targets. By introducing the novel STexS II, omicron variants of SARS-CoV-2 were able to be successfully detected in high concentrations of normal genes.

Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection

Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.

Virulence determinants and genotypes of <i>Helicobacter pylori</i> clinical isolates

Background. H. pylori is the principal causative agent of gastroduodenal disorders in humans. The development and severity of lesions in infected individuals depend on the virulence of H. pylori strains.
 Aims: Detection of virulence determinants and comparative analysis of H. pylori genotypes in patients with chronic gastritis (CG) and duodenal ulcer (DU).
 Materials and methods. The 53 H. pylori strains were isolated in St. Petersburg from patients with CG (n = 34) and DU (n = 19). The genetic determinants of virulence cagA, iceA, vacA and H. pylori genotypes in patients with CG and UC were determined using the standard PCR method.
 Results. The cagA gene was found in 64.1% of H. pylori strains. The proportions of cagA+ isolates from patients with CG and DU was 55.8% (15/34) and 78.9% (15/19), respectively (p 0.05). The iceA1 allele of H. pylori was detected in 47.4% of patients with DU, the iceA2 in 47.1% of patients with CG (p 0.05). The vacAs1 allele was significantly dominant in patients with DU 94.7% versus 70.6% in CG (p 0.05). No significant difference in vacA m1 and m2 alleles was found in H. pylori from different groups of patients (p 0.05). All cagA+ strains were carriers of the vacA s1 allele. The vast majority of strains (10 out of 11) of the cagA/vacAs2 genotype were isolated from patients with CG.
 Conclusion. The significant association between vacAs1, vacAs2 allelic variants, as well as vacA s1/m2, vacA s2/m2 genotypes of the pathogen and severity of clinical manifestations of H. pylori infection has been established in our study. The vacAs1 and vacA s1/m2 genotypes of the pathogen are associated with duodenal ulcer.

Опыт использования синдромальных подходов к диагностике респираторных инфекций

Irrational or excessive use of antibiotics, the absence or insufficient use of rapid diagnostics tests at the point of care ("POC"), as well as insufficient infection control are key factors in the development of multi- and pan-resistance to antibiotics. The article presents the clinical experience of using the syndromic approach Patients and methods. The work was carried out on the basis of the Morozovskaya State children's hospital from February to June 2022 in accordance with the standards of medical care for patients with acute respiratory infections using multiplex PCR test (mPCR) – BioFire FilmArray respiratory panel (BioFire RP). The study included inpatient children (n = 40) with symptoms of acute respiratory infections and respiratory failure (DN) of different severity. Results. The analysis of the nosological structure showed that acute obstructive bronchitis/acute bronchitis (J20.9) was found in 47.5%, pneumonia (J18.9) in 20% of children, acute bronchiolitis (J21.9) at 7.5%, other viral lesions of the respiratory tract (J00-J06) – 25%. The standard PCR ARVI panel identified pathogens in 45% (n = 18), the BioFire RP panel in 55% (n = 22) of patients (p = 0.503). An important indicator is the speed of obtaining results. The median time from the moment of taking the smear to the moment of transmitting the result to the physician was 2.56 hours (CI: 1.91–3.62) when examined using the BioFire RP panel compared with 28.4 hours (CI: 9.64–33) when using standard PCR diagnostics for respiratory viruses (p < 0.001). Conclusion. Methods of rapid molecular diagnostics can reduce the time of hospitalization of patients. In the study, the duration of hospitalization is 1 day shorter compared to the analysis of retrospective indicators of hospitalization terms. This is due to the ability to quickly identify the agent of respiratory infection, which in turn affects the tactics of treatment, optimizes the appointment of antibiotics, which affects to the duration of hospitalization. Key words: respiratory infection, BioFire, PCR, virus, rapid diagnosis, etiotropic therapy

Design and validation of multiplex polymerase chain reaction as a diagnostic tool for Plasmodium species.

The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations. The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus. The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR. The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.

Low Volume STR Amplification Options: Coupling with Standard or Fast PCR, Traditional or Normalized DNA Extraction, and/or Traditional or Alternative Capillary Electrophoresis.

STR amplification leads directly to profile development, which is also impacted by DNA extraction and capillary electrophoresis detection. Amplification for forensic human identification purposes is inherently a costly process; reduced volume reactions have long been an effective cost-savings measure. Processing known, high-quality, single-source DNA samples (i.e., buccal samples) allows for the use of even lower reaction volumes. This chapter provides examples of 3μL and 6μL reactions for a variety of commercial amplification kits for use with buccal samples, including standard and fast PCR using KAPA2G™ Multiplex Mix. These reactions can be utilized with traditional DNA extracts or those obtained from a normalized extraction with the ChargeSwitch® Forensic DNA Purification Kit. They can be detected via traditional capillary electrophoresis using POP-4™ polymer and a 36cm array, or an alternative method using POP-6™ polymer and a 22cm array on the 3130 series Genetic Analyzer instruments. This chapter also includes protocols for the normalized extraction and alternative detection method.

Reverse transcription-free digital-quantitative-PCR for microRNA analysis.

MicroRNAs (miRNAs) are non-coding RNA sequences that regulate many biological processes and have become central targets of biomedical research. However, their naturally low abundances in biological samples necessitates the development of sensitive analytical techniques to conduct routine miRNA measurements in research laboratories. Digital PCR has the potential to meet this need because of its single-molecule detection capabilities, but PCR analyses of miRNAs are slowed by the ligation and reverse transcription steps first required to prepare samples. This report describes the development of a method to rapidly quantify miRNA in digital microwell arrays using base-stacking digital-quantitative-PCR (BS-dqPCR). BS-dqPCR expedites miRNA measurements by eliminating the need for ligation and reverse transcription steps, which reduces the time and cost compared to conventional miRNA PCR analyses. Under standard PCR thermocycling conditions, digital signals from miRNA samples were lower than expected, while signals from blanks were high. Therefore, a novel asymmetric thermocycling program was developed that maximized on-target signal from miRNA while minimizing non-specific amplification. The analytical response of BS-dqPCR was then evaluated over a range of miRNA concentrations. The digital PCR dimension increased in signal with increasing miRNA copy numbers. When the digital signal saturated, the quantitative PCR dimension readily discerned miRNA copy number differences. Overall, BS-dqPCR provides rapid, high-sensitivity measurements of miRNA over a wide dynamic range, which demonstrates its utility for routine miRNA analyses.

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Colletotrichum abscissum: Detection on symptomless citrus leaves and symptomatic citrus flowers using high‐resolution melting analysis

AbstractHigh‐resolution melting (HRM) analysis has been recently applied for the diagnosis of plant diseases. This cost‐effective method is advantageous over standard PCR as it does not require DNA band visualization or the use of probes necessary for quantitative PCR. Colletotrichum abscissum causes postbloom fruit drop of citrus (PFD) and survives asymptomatically on vegetative tissue. Quiescent appressoria on citrus leaves are the primary source of inoculum between flowering periods. Early PFD symptoms may be misidentified as physical damage on citrus flowers. Our objectives were to validate an HRM‐based method to rapidly identify C. abscissum on citrus leaves and flowers. We screened seven previously published primer pairs and concluded that the most effective set of primers was CaITS‐F and CaITS‐R815. We evaluated three different DNA extraction methods, two with a purification step (DNeasy Plant Mini and PowerSoil kits) and a faster method without a purification step (buffer GEB2). We inoculated citrus leaves with C. abscissum conidial suspensions of varying concentrations and HRM analysis detected as few as 200 C. abscissum spores using DNeasy Plant Mini or DNeasy PowerSoil kits for DNA extraction. As expected, samples extracted with GEB2 buffer reduced HRM sensitivity. We further collected field samples from areas with high and low C. abscissum inoculum levels. The HRM method could detect the pathogen in high inoculum areas from both leaves and flowers regardless of the extraction method used. In summary, we describe a new molecular diagnostic tool to detect C. abscissum on citrus during its asymptomatic and symptomatic stages.

Emergence of Chronic Myeloid Leukemia in Patients with Myeloproliferative Neoplasms: A Single-Institution Experience

Introduction: Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 translocation and is typically distinct from other myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Concurrent diagnosis of CML and MF is uncommon and infrequently described in case reports.1 Moreover, very few cases have been described of CML occurring after an established diagnosis of MPN.2-3 Due to the rarity of concurrent CML and MPN, management strategies in this population are not defined. In addition, the latency time from the emergence of BCR-ABL1 positive clones to a clinical diagnosis of CML in this setting is largely unknown. To address these questions, we interrogated our institutional experience and retrospectively analyzed available specimens. Methods: We collected information on patients seen at Massachusetts General Hospital (MGH) from January 1 2012 to January 1 2022 for CML occurring after an established diagnosis of MPN. Available stored samples were retrospectively tested for BCR-ABL1 with a fusion transcript assay (MGH Center for Integrated Diagnostics, Boston, MA). Results: We identified four patients with CML preceded by MPN (Table 1). Three patients had prior MF, diagnosed 4.3-7.6 years before the onset of CML; two of these had post-ET MF. Patient #4 had ET diagnosed 4 years prior to onset of CML, then went on to develop MF 4.9 years after CML diagnosis. None of the patients were on JAK inhibitors, hydroxyurea or interferon at the time of CML diagnosis. All patients had a leukocytosis with characteristic features on the differential prompting evaluation for CML. The median white blood cell count was 45.83 x 10^9/L (range 27.49-121.02). All four patients had an elevated basophil count (median 2.99 x 10^9/L, range 1.1-22.99) and two had an elevated eosinophil count (median 1.11 x 10^9/L, range 0.55-44.17). Regarding MPN driver mutations, one patient had a JAK2 V617F, one patient had a CALR exon 9 deletion, and two patients had no identifiable mutations on extended next-generation sequencing. For CML treatment, three patients received dasatinib (two with 100 mg daily, one with 50 mg daily), whereas one received nilotinib (150 mg twice daily). Two achieved a major molecular response (MMR) (at 7 and 8 months); one had an early molecular response at 6 months (last follow-up); one (with e13-a3 transcript) could not be quantified on a standard PCR assay but achieved a complete cytogenetic response by 4 months. At the time of last follow-up, two patients were in MMR on tyrosine kinase inhibitors (TKI), whereas two underwent allogeneic hematopoietic cell transplantation (alloHCT), primarily for progression of MF. We retrospectively interrogated available samples for evidence of the BCR-ABL1 translocation prior to a clinical diagnosis of CML. Patient #2 was followed at MGH for MF and thus had available samples. We identified BCR-ABL1 transcripts in the peripheral blood three months prior to his clinical diagnosis of CML (unable to be quantified due to e13-a3 transcript); there were no available samples from earlier in his disease course for testing (Figure 1). Discussion: CML can emerge in patients with MPN. Herein, we describe four cases of this phenomenon. Signs of CML evolution included marked leukocytosis, as well as basophilia and eosinophilia. Despite underlying MF, three patients tolerated full-dose TKI and achieved molecular responses early in their disease course. However, MPN progression remained a significant challenge, with two patients ultimately requiring an alloHCT. Of note, we found evidence of BCR-ABL1 in the peripheral blood three months prior to clinically apparent CML in one patient. Experiments using single cell techniques in patients with concurrent MPN and CML would provide insights into the clonal architecture and are in planning stages.

A detailed review of contrast-enhanced fluorescence magnetic resonance imaging techniques for earlier prediction and easy detection of COVID-19

ABSTRACT Omicron, a novel coronavirus disease (COVID-19) variation, just seems to contain hundreds of modifications, with no transition sequences found in their ribonucleic acid (RNA). Omicron has a mutation termed E484A, as well as polymorphisms in the sites where it can attack two kinds of antibodies at one time. Since the nucleotide was highly altered, the well-known gold standard PCR test failed to identify these variants in post-covid patients. Computer tomography (CT) and Lung-X-Ray tests are a little more effective than the real-time reverse transcription–polymerase chain reaction (real-time RT – PCR) test in the diagnosis of coronavirus transmission (usually after 2 weeks). So, it is a need of the hour to define a test that could identify the symptoms, and extend the impact of COVID-19 in the pre-and post-COVID-19 cases. The in vivo MRI and the advanced fluorescent magnetic resonance imaging (F-MRI) diagnostic test for the lungs were recently revamped to identify the essential symbols of viral pneumonia, and pathological alterations in lung nodules, and made it easy to identify the seriousness of respiratory problems. The present work aims to review F-MRI techniques for earlier prediction and easy detection of COVID-19.

Investigating transmission of SARS-CoV-2 using novel face mask sampling: a protocol for an observational prospective study of index cases and their contacts in a congregate setting

IntroductionThis study aims to measure how transmission of SARS-CoV-2 occurs in communities and to identify conditions that lend to increased transmission focusing on congregate situations. We will measure SARS-CoV-2 in...

A rapidDNAextraction method for the detection ofClavibacter sepedonicusandRalstonia solanacearumin potato samples in a multiplexPCRtest

AbstractA method is described that generates DNA extracts suitable for the detection ofClavibacter sepedonicusandRalstonia solanacearumin potato samples, without the need for laborious, time‐consuming extraction protocols or expensive kits and laboratory equipment. Furthermore, a multiplex test is presented that allows the simultaneous detection of both pathogens in one standard PCR reaction. The methods described here allowed the detection of both bacteria in cell densities of 103cells per mL of plant extract, thus fulfilling the demands of official testing schemes, e.g. in the European Union. High potato starch content did not affect analytical sensitivity, and high concentrations of one pathogen did not impede the detection of the other. This method might be useful for all laboratories worldwide carrying out medium‐ to large‐scale potato testing for bacteria, and also seems to be suitable for the detection of virus diseases relevant for seed potato certification.

Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective.

The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.

Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida.

Aeromonas salmonicida is the pathogen underlying furunculosis, causing a septicemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so a rapid and sensitive approach is needed. Herein, a recombinase polymerase amplification EXO (RPA-EXO) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA-EXO and RPA-LFD primer sets were devised based on the conserved fragment sequence of the vapA gene. Then, RPA-EXO and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA-EXO method was capable of testing A. salmonicida within 10min, and the RPA-LFD method could detect A. salmonicida in only 5min. The RPA-EXO and RPA-LFD methods exhibited high specificity with no cross-reaction with other strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1 × 106 to 1 × 10-1 copies/μL. The detection limit of RPA-EXO was 1 × 102 copies/μL, and the detection limit of RPA-LFD was 1 copy/μL. For spiked turbot tissue samples, the sensitivity detection of A. salmonicida was 1.2 × 101CFU/mL and 1.2CFU/mL by RPA-EXO and RPA-LFD, respectively. In comparative analyses of clinical samples, the diagnostic results of RPA-EXO and RPA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our RPA-EXO and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.

Detection of Tick-Borne Pathogens in Ticks from Cattle in Western Highlands of Cameroon.

This study aimed to detect and identify microorganisms in ticks collected in the Western Highlands of Cameroon. Quantitative real-time and standard PCR assays, coupled with sequencing, were used. A total of 944 ticks collected from cattle in five distinct sites in Cameroon were selected for the analyses. They belonged to five genera (Amblyomma, Hyalomma, Rhipicephalus, Haemaphysalis, and Ixodes) and twelve species. Real-time PCR revealed that 23% (n = 218) of the ticks were positive for Rickettsia spp., 15% (n = 141) for bacteria of the Anaplasmataceae family, 3% (n = 29) for Piroplasmida, 0.5% (n = 5) for Coxiella burnetii, 0.4% (n = 4) for Borrelia spp., and 0.2% (n = 2) for Bartonella spp. The co-infection rate (3.4%, n = 32) involved mainly Rickettsia spp. and Anaplasmataceae. Of the Rickettsia spp. positive ticks, the targeted PCR and sequencing yielded Rickettsia africae (78.9%), Rickettsia aeschlimannii (6.4%), Rickettsia massiliae (7.8%), Candidatus Rickettsia barbariae (0.9%), and Rickettsia sp. (0.9%). Anaplasmataceae included Anaplasma marginale (4.3%), Anaplasma platys (1.4%), Anaplasma centrale (0.7%), Ehrlichia ruminantium (0.7%), Wolbachia sp., Candidatus Ehrlichia rustica (13.5%), Candidatus Ehrlichia urmitei (7%), and an uncultured Ehrlichia sp. (4.2%). Borrelia theileri was identified in one Rhipicephalus microplus tick. Unfortunately, Piroplasmida could not be identified to the species level. This study demonstrates that in Cameroon, ticks harbour a wide variety of microorganisms and present a risk of zoonotic diseases.

Cultivable microbiota of Proteus anguinus from underground habitats and animals accidently washed to the surface in Croatia

Proteus anguinus (olm) is an amphibian endemic to underground cave systems of the Dinaric karst of Central Europe, adapted to a life in complete darkness. Recent studies have demonstrated the global decline of amphibians due to poorly understood microbiological diseases, emphasizing just how little is known about the natural microflora of amphibians. Not much is known about the olm’s microbiota in their cave habitats, and nothing is known about the microbiology of animals washed to the surface. This observational study describes the microbiological analysis of the skin, cloaca and oral cavity of six olms collected from their cave habitats, and 16 collected after being washed out by flows from two springs, to learn more about the olm’s normal microbiota and possible changes after contact with the different environmental conditions on the surface. Standard microbiological procedures, MALDI-TOF and Real-Time PCR were used for microbiological species identification. All animals tested negative for Ranavirus, Batrachochytrium dendrobatidis, Batrachochytrium salamandrivorans and Chlamydia spp. The most abundant fungi isolated were Penicillium spp. and Cladosporium spp. The washed out animals showed a higher diversity of bacterial flora than those from cave habitats, with Acinetobacter johnsonii, Aeromonas hydrophila, Bacillus spp. and Janthinobacterium lividum as the most frequently identified isolates.

Simple and low-cost nucleic acid extraction methods for detection of SARS-CoV2 in self-collected saliva and dry oral swabs

BackgroundOngoing need of alternative strategies for SARS-CoV-2 detection is undeniable. Self-collected samples without viral transport media (VTM), coupled with simple nucleic acid extraction methods for SARS-CoV-2 PCR are beneficial. ObjectivesTo evaluate results of SARS-CoV-2 PCR using simple nucleic acid extraction methods from self -collected saliva and oral swabs without VTM. MethodsA cross-sectional single-centre study was conducted on 125 participants (101 SARS-CoV-2 positive cases and 24 controls). PCR was performed following five simple nucleic acid extraction methods on self -collect saliva and oral swabs without VTM and results were compared with gold standard PCR. For saliva, kit-based extraction (SKE), Proteinase K and Heat extraction (SPHE), only Heat extraction (SHE) methods and for dry oral swabs, Proteinase K and Heat extraction (DPHE) and only Heat extraction (DHE) was performed. ResultsSARS-CoV-2 was detected in self-collected saliva and oral swabs. 93.07% were correctly classified as positive by SKE, 69.31% by SHE, 67.33% by SPHE, 67.33% by DPHE and 55.45% by DHE. Discriminant power of SKE was significantly higher than other methods (p-value < 0.001) with good- fair agreement of alternate extraction methods against gold standard. ConclusionCombination of self-collected saliva/ oral-swab without VTM and alternative RNA extraction methods offer a simplified, economical substitute strategy for SARS-CoV-2 detection.