PCR Run Research Articles

A retrospective study of vector borne disease prevalence among anemic dogs in North Carolina.

Anemia is an important cause of morbidity and mortality in dogs. Further understanding of the prevalence of vector borne diseases (VBD) in anemic dogs is needed. The objective of this retrospective study was to describe the rate of exposure to or infection with VBD among anemic dogs presented to a teaching hospital in North Carolina and to further characterize the anemia in dogs with VBD exposure. A total of 597 anemic dogs that were concurrently tested for VBD were examined at a referral veterinary hospital between January 2012 and December 2018. Retrospective descriptive study. Demographic, clinicopathologic, and VBD testing data were obtained from medical records. Of the 597 anemic dogs examined, 180 (30.15%; 95% CI: 26.49-34.01%) tested positive for one or more VBD. There was no difference in the severity of anemia or the proportion of dogs displaying a regenerative anemia between dogs testing positive and negative for VBD. A large proportion of anemic dogs from this region test positive for exposure to or infection with VBD. Our study supported the use of PCR and serology run in parallel to maximize the chance of detecting exposure to or infection with VBD compared to either serology or PCR alone. At this time, it is unknown whether infection with VBD contributed to the development of anemia in these patients. However, given the prevalence of VBD exposure in anemic dogs, testing for VBD in anemic patients from this region of the United States is warranted.

A New qPCR Assay for the Rapid Diagnosis of Anthonomus grandis Subspecies.

Rapid and accurate identification of Anthonomus grandis subspecies is crucial for effective management and eradication. Current diagnostic methods have limitations in terms of time to diagnosis (up to seven days) and can yield ambiguous results. Here, we present the validation of a custom TaqMan SNP Genotyping Assay for the rapid and accurate identification of A. grandis grandis (boll weevil) and A. g. thurberiae (thurberia weevil) subspecies. To validate the assay, we conducted three main experiments: (1) a sensitivity test to determine the DNA concentration range at which the assay performs, (2) a non-target specificity test to ensure no amplification in non-target weevils (false positives), and (3) an accuracy test comparing the results of the new assay to previously established methods. These experiments were carried out in parallel at three independent facilities to confirm the robustness of the assay to variations in equipment and personnel. We used DNA samples from various sources, including field-collected specimens, museum specimens, and previously isolated DNA. The assay demonstrated high sensitivity (PCR success with ≥0.05 ng/µL DNA template), specificity (0.02 false positive rate), and accuracy (97.7%) in diagnosing boll weevil and thurberia weevil subspecies. The entire workflow, including DNA extraction, assay preparation, PCR run time, and data analysis, can be completed within a single workday (7-9 h) by a single technician. The deployment of this assay as a diagnostic tool could benefit boll weevil management and eradication programs by enabling same-day diagnosis of trap-captured or intercepted weevil specimens. Furthermore, it offers a more reliable method for identifying unknown specimens, contributing to the overall effectiveness of boll weevil research and control efforts.

Development and Application of a Triplex PCR Assay for Detection of Koi Herpes Virus (KHV) Gene Infected Common Carp (Cyprinus carpio L.)

Koi herpesvirus (KHV) has been responsible for worldwide KHV disease (KHVD) outbreaks, resulting in high mortality rates, and has been reported in Cyprinus carpio cultivation. A Triplex Polymerase Chain Reaction (T-PCR) was developed for the detection of KHV gene to improve the diagnosis of KHV infection. Three sets of primers were designed targeting specific sequences of KHV gene which were frequently used in PCR assay for detecting KHV (modifications primer by Yuasa,290 bp; Gilad 484 bp, and TK, 409 bp) in once PCR running. The validity test was conducted by verified using DNA sample of Aeromonas hydrophila. Sensitivity test of singleplex and Triplex conducted by dilution. The result showed that T-PCR method using these pairs of primers is at 55 annealing temperature with consistency of T-PCR method has been repeated in triplicates, and with the same results. The validity test found that no band detected with A. hydrophila. This Triplex PCR-based assay may have useful for the clinical diagnosis tool of KHV disease.

Carbon-based biosensors: Next-generation diagnostic tool for target-specific detection of SARS-CoV-2 (COVID-19)

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) was declared a global pandemic in 2020. Having rapidly spread around the globe, with the emergence of new variants, there is a crucial need to develop diagnostic kits for its rapid detection. Since it validated accuracy and reliability, the reverse transcription polymerase chain reaction (RT-PCR) test has been declared the gold standard for disease detection. However, despite its reliability, the requirement of specialized facilities, reagents, and duration of a PCR run limits its usage for rapid detection. There is thus a continuous increase in the design and development of rapid, point-of-care (PoC), and cost-effective diagnostic kits. In this review, we discuss the potential of carbon-based biosensors for target-specific detection of coronavirus disease 19 (COVID-19) and present an overview of investigation within the timeframe of the last four years (2019–2022), which have developed novel platforms using carbon nanomaterial-based approaches for viral detection. The approaches discussed offer rapid, accurate, and cost-effective strategies for COVID-19 detection for healthcare personnel and research workers.

Point-of-Care Testing for Sensitive Detection of the African Swine Fever Virus Genome.

African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.

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.

PB2254: ASSOCIATION BETWEEN TNF-ALPHA LEVELS AND TNF-ALPHA 238 ALLELES POLYMORPHISMS AND SEVERITY OF ANEMIA AMONG SUDANESE CHILDREN WITH FALCIPARUM MALARIA

Background: Falciparum malaria is the most leading cause of mortality and morbidity in the world especially in Africa among infants and young children due to severe malaria and malaria anemia. TNF-α is plays a central role in malaria pathogenicity either in the cure or complication of malaria. TNF-α levels overproduction and promoter polymorphisms at TNF-α 238 alleles may play a central role in reduced red cell production and malaria-related anemia through suppression of bone marrow erythropoiesis and dyserythropoiesis. Aims: The purpose of this study was to evaluate the TNF-α 238 alleles polymorphism and its association with TNF-α levels in the severe malaria anemia among Sudanese children in Gezira State. Methods: A cross-sectional hospital-based study was included 200 Sudanese children (mean age 8.43 ± 3.80 years; 53% boys; 47% girls). 100 children with severe falciparum malaria (mean age 8.63 ± 3.40 years; 61% boys; 39% girls) and 100 children with uncomplicated falciparum malaria (mean age 8.83 ± 4.20 years; 45% boys; 55% girls). RBCs parameters were determined using the Sysmex XP 300 N automated hematology analyzer. ELISA was further processed for TNF-α level using Human TNF-α ELISA MAX™ Deluxe Sets. DNA extraction was done using G-DEX™IIb Genomic DNA Extraction Kit. PCR and gel running system were used for detecting TNF-α 238 alleles polymorphism (A allele/ G allele). Obtained data were analyzed by SPSS (V 20.0) and Stat disk (V 13.0). Results: TNF-α 238A allele was a common allele (66.8%) among Sudanese children with falciparum malaria, and the G allele was rare (33.2%). While TNF-α 238 GA, AA, and GG account for (54.5, 39.5, and 6% respectively). Falciparum malaria-related anemia accounted for 32%, commonly in SM (55%) compared to UM (9%) giving highly statistically significant differences in TNF-α levels between falciparum malaria-related anemia and falciparum malaria without anemia (P value 0.000). Otherwise, The average of TNF-α levels in mild, moderate, and severe anemia were (190.75 ± 102.55, 189.70 ± 80.35, and 299.75 ± 82.27 pg/ml respectively) giving highly significant differences and strongly significant positive correlated with the severity of anemia (r + 0.309; P value 0.000). TNF-α 238 GA, AA, and GG account for (58, 36, and 6% respectively) in UM; while (51, 43, and 6% respectively) in SM. The TNF-α 238 A allele accounts for 83.6% of malaria anemia (P value 0.000) and 100% severe anemia (P value 0.000); children who have TNF-α 238 A allele, they have ability to acquire falciparum malaria-related anemia with susceptibility to 3.13 folds risk. Summary/Conclusion: Overproduction of TNF-α is essential for the elimination and clearance of falciparum parasite but may be associated with severity of malaria and malaria anemia. Overproduction of TNF-α in children with TNF-α 238 A allele may result in falciparum malaria-related anemia among children (mainly severe anemia). The findings of this study will assist clinicians in diagnosing and better managing severe malaria cases.

A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

Development of a triplex real-time PCR system for the differentiation between Brassica plant species

Reliable monitoring of the authenticity of food and feed depends on correct analytical methods. One source of error might be false positive results. This might be especially the case with closely related or interrelated species. One such genus is Brassica which consists of the three diploid species Brassica rapa (A genome), B. nigra (B genome), and B. oleracea (C genome) and the three allopolyploid plant species B. juncea (AB), B. napus (AC), and B. carinata (BC). All six Brassica plant species have in common that they are used for food and feed worldwide. To be able to discriminate between all six of them in one real-time PCR run, a triplex real-time PCR system was established which identifies the three genomes (A, B, or C). The efficiency, R2, and LOD values are within the expected range. The applicability of monitoring the authenticity of processed food was shown by analyzing emulsified sausages with 0.0005–0.1% (w/w) rapeseed protein, 2% broccoli, or 0.1% brown mustard seeds. The detection and differentiation were possible at all concentration levels.

An analysis of the standard curve parameters of cytomegalovirus, BK virus and hepatitis B virus quantitative polymerase chain reaction from a clinical virology laboratory in eastern India

PurposeQuantitative real-time polymerase chain reactions (qPCRs) are important for accurate detection of nucleic acid target including that for viral load determination. Assessment of the quality of a PCR run is essential for quality control, diagnostics and research. In order to reduce subjectivity qPCR standard curves are accompanied with parametric values for slope, Y- intercept, correlation coefficient (R2) and PCR efficiency. In this study the performance of three qPCR assays-cytomegalovirus, hepatitis B virus and BK virus-with respect to standard curve parameters-slope, Y intercept, R2 and efficiency were examined. MethodsUsing ideal values (Slope (minus 3.32); Y intercept ​= ​the number of PCR cycles; R2 ​= ​1 and efficiency ​= ​100%) we estimated the intra-assay variability (range) and deviation from ideal parameters (Δ). We also calculated the standard deviation (SD) and coefficient of variation (CV) for each of these parameters. We have evaluated the quality of each of the three viral load assays (CMV, HBV, BKV) using these statistical approach. ResultsWe found lab developed tests (CMV) to have least deviation from ideal Y intercept (limit of detection); however, commercial kit based assays had better linearity (scatter plot correlation between amplification factor and PCR efficiency). Using a scatter plot for the three assays we found the correlation with calculated amplification factor and PCR efficiency was most linear in case of BKV (0.9974), closely followed by the HBV assay (R2 ​= ​0.9968). Although the CMV quantitative standards were least linear (0.868), the CV (coefficient of variation) was also the least in case of the CMV assay. ConclusionThe study highlights an objective way of assessing qPCR assay quality and demonstrates a method to compare assays, validate tests and perform quality control.

Comparative Evaluation of the SMARTCHEK Salmonella Species Detection Kit for GENECHECKER UF-300 Real-Time PCR System with Rapi:chipTM and Rapi:DirectTM Lysis Buffer for Select Foods: AOAC Performance Tested MethodSM 032101

The candidate method, SMARTCHEK Salmonella spp. Detection Kit assay is designed for rapid detection of Salmonella spp. in select food matrixes, for use in conjunction with the Genesystems Rapi:chipTM and GENECHECKER UF-300 real-time PCR system. The independent laboratory evaluation consisted of an inclusivity and exclusivity study, product consistency study, robustness study, stability study, and a matrix study. The paired matrix study tested romaine lettuce, peanut butter, liquid whole egg, chicken carcass rinsate, and raw chicken. The robustness study evaluated the method by changing three method parameters. The accelerated stability and product consistency study was conducted at 8, 16, 24, 32, and 40 days. The candidate method was compared to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook 4.10 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Siluriformes (Fish) Products and Carcass and Environmental Sponges and to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 5: Salmonella following a paired study design. All 108 Salmonella isolates were detected in all three broths tested for inclusivity, while all 35 non-Salmonella isolates were correctly excluded. In the matrix study, the candidate method showed no significant differences between confirmed candidate method results and reference method results for all food matrixes evaluated. The candidate method is an effective method for the qualitative detection of Salmonella spp. in select food matrixes. The SMARTCHEK Salmonella spp. Detection Kit assay allows for fast, reliable detection of Salmonella spp. with results obtained after 24 h of incubation and a PCR run time of only 35 min.

Genetic Polymorphism of Tumor Necrosis Factor-Alpha, Interferon-Gamma and Interleukin-10 and Association With Risk of Mycobacterium Tuberculosis Infection.

Background. Mycobacterium tuberculosis has become the leading cause of morbidity and death in humans worldwide. Thus, genetic variability of the host plays a major role in human susceptibility to the pathogen, among others. Therefore, the objective of this finding was to assess the association of genetic polymorphisms of cytokines with tuberculosis infection.Method.A cross-sectional study was conducted between January and May 2018. Five ml of whole blood was collected and extracted the genomic DNA through simple salting out method. The patterns of genetic polymorphism were determined by amplification refractory method PCR using specific primers. Finally, the PCR run on electrophoresis of agarose gel and the band was visualized under UV light. A logistical regression model has been adapted to assess the association of genetic polymorphisms with tubercular infection. In order to determine the association between the explanatory and outcome variable, the odds ratio with 95% CI was calculated. P < 0.05 is a statistically significant value.Result.In present study, the frequency of TNF-α -308 G allele and GG genotype OR (95% CI)= 0.20 (0.11-0.37), and OR (95% CI)= 0.29 (0.18-0.46)), respectively) and IFN-γ +874 A allele and AA genotype OR (95% CI)= 3.80 (2.11-6.86) and (OR (95% CI)= 1.61(1.13-2.28), respectively) were significantly associated with tuberculosis incidence. In contrast, there is no significant correlation between IL-10 -1082 A and AA of allele and genotype, respectively in tuberculosis patients (p > 0.05) was evident.Conclusion.From our finding, the genetic variability of TNF-α -308 A and IFN-γ +874 alleles are the potent host genetic risk factors associated with tuberculosis infection.

Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (&gt;30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

Uninterruptible Power Supply Improves Precision of Telomere Length Measurement via qPCR

Epidemiological literature has produced robust associations between telomere length (TL) and health, wherein individuals with shorter TL are at increased risk for chronic disease and death. Even so, technical challenges associated with TL measurement have led some to question their utility as a biomarker of aging. Several pre-analytical factors influence TL assessment via qPCR including tissue source, sample storage, and DNA extraction method. Additional work has investigated how conditions within the PCR run (e.g. mastermix reagents) influences precision and reproducibility of TL measurements. However, the impact of power supply remains unclear. Momentary fluctuations in power supply can affect the functioning of high-performance electronics, including real-time thermocylers. These fluctuations can be mitigated by using an uninterruptible power source (UPS), an electronic apparatus capable of supplying constant, uninterrupted voltage to high-performance electronics. The current study investigated how using a UPS influenced TL assessment via qPCR. Standard deviation and coefficient of variation across replicates were compared for samples assessed with or without the use of a UPS. Samples run with a UPS had significantly lower standard deviation (p<0.001) and coefficient of variation (p=0.002) than those run without a UPS. Notably, neither the efficiency of exponential amplification (p=0.674) nor the standard curve R2 (p=0.638) varied as a function of UPS status. Thus, UPS status decreases variability within sample replicates while maintaining the overall quality of the qPCR assay. *The work presented was supported by the Telomere Research Network, an NIH-sponsored working-group recently established to coordinate best practices for measuring telomere length in population-based research.

Development of a fully automated high throughput PCR for the detection of SARS-CoV-2: The need for speed

ABSTRACT Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84–97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.

Abstract A118: Optimization of end point PCR for the determination of ESR1, PGR, and ERBB2 expression level in resource-limited settings: Ethiopian context

Abstract Background: Breast cancer is becoming a major public health problem in developing countries. Accumulated evidence noted that the majority of breast cancer patients are in a younger age group. Moreover, most breast cancer patients appear in health institutions at late stages of the disease, which compromises the clinical outcome and disease management. With regard breast cancer, it solely depends on pathologic and clinical diagnoses. The characterization of hormone receptors and HER-2 status using immunohistochemistry (IHC) has been introduced in Ethiopia; however, there is critical problem with the provision of requirements for the test. As a result, the development and establishment of a rapid, reproducible, and alternative diagnostic method in resource limited setting is essential. The current research work aims to optimize endpoint PCR as a tool for determination of ESR1, PGR, and ERBB2 expression level using breast cancer specimen in Ethiopian patients. Furthermore, the project work will be extended to implement the method into clinical practice in the Ethiopian settings. Methods: The experiments were started with known ESR1, PGR, and ERBB2 RNA expression in different breast cancer cell lines such as MCF-7, MDA-MB-231, BT-20, and SKBR-3 .cDNA from the breast cancer cell lines and FFPE derived-RNAof BC lesions was synthesized using BiozymcDNA synthesis kit. Variable concentrations (25ng, 50ng, and 100ng) of breast cancer cell lines and FFPE-derived total RNA were used for ESR1, PGR, and ERBB2 mRNA transcript detection using the corresponding primer pairs of each gene. GAPDH served as a reference gene. The following PCR program was applied: denaturation 950c (1min), 620c annealing temperature (20sec), 720c synthesis/extension temperature (20sec) for 35 cycles. The experiments were performed by including non-template controls in each PCR run. The PCR products were subjected to gel electrophoresis using SERVA agarose standard electro endosmosis (EEO)followed by documentation with the Image QuantTM LAS4000. Results: Multiple PCR experiments were carried out with different counts of PCR cycles, with and without extension, with various RNA concentrations and using breast cancer cell lines and FFPE-derived RNA. With the PCR conditions, testing of ESR1, PGR, and ERBB2 expression was possible. In particular, the MCF-7, MDA-MB-231 cell lines PCR run produced concordant data with IHC. However, some BC cell lines (BT-20 and SKBR-3) known as negative for the respective hormone receptor showed discordant results. Conclusion: In summary, the determination of the expression of ESR1, PGR and ERBB2 level in BC was successful. However, it requires further tests using different PCR conditions and a large set of known human breast tissues. From the experiment, we have learnt that endpoint PCR based ESR1, PGR, and ERBB2 detection can be used as an alternative method for better BC receptor diagnosis, treatment plan, and management in resource-limited countries. Citation Format: Zelalem Desalegn Woldesonbet, Martina Vetter, Meron Yohannes Nigussie, Tamrat Abebe Zeleke, Yonas Bekuretsion, Mahlet Arayselassie, Mathewos Assefa, Abebe Bekele, Endale Anberber1, Claudia Wickenhauser, Eva J Kantelhardt, Jürgen Bukur, Seliger Barbara. Optimization of end point PCR for the determination of ESR1, PGR, and ERBB2 expression level in resource-limited settings: Ethiopian context [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A118.

DNA barcoding for the identification of mold species in bakery plants and products

Mold identification at the species level in environmental samples is a major challenge. Molecular techniques have been widely used for fungal classification, but as most primers are genus-specific, it is laborious to identify unknown samples. In this study, a PCR-based method for the identification of mold at the species level was developed. Therefore, common sequencing primers and combinations of them, targeting specific DNA regions, were tested. Here we present a combination of eight primer pairs to identify mold within a single PCR run. The approach correctly identified mold of unknown species from samples taken at a local bakery, including Penicillium chrysogenum, Penicillium citrinum, Cladosporium sphaerospermum, Paecilomyces formosus, Rhizopus oryzae and Aspergillus niger. Results obtained from the PCR method were successfully validated by chromatographic mycotoxin and microscopy analysis. Findings highlight DNA barcoding as an appropriate tool for mold identification; however, its efficacy is essentially dependent on DNA quality and primer selection.

Early detection of Cryphonectria parasitica by real-time PCR

The development and validation of a real-time PCR method for the detection of Cryphonectria parasitica in bark tissues is described. The selected region in the genome was a fragment of the internal transcribed spacer region. The DNA extraction and PCR conditions were optimized to be routinely applicable to fresh and dried bark. The sensitivity of the assay allowed the detection of 2 fg of genomic DNA, equivalent to one spore of the pathogen. There was no cross-reaction with closely related Cryphonectria species. The use of droplet digital PCR (ddPCR) confirmed the high sensitivity of the real-time PCR method, and its capacity to be used as an early detection method. A survey was conducted in Belgium on chestnut trees displaying cankers using isolation and the real-time PCR method. Both methods provided the same results for 83% of the samples (either negative or positive results). For the remaining samples, C. parasitica was not isolated, while amplifications close to the end of the PCR run (resulting in late Cycle threshold (Ct) values) were observed with the real-time PCR. Some of these samples were collected in stands where C. parasitica had already been detected on other chestnut trees, or in sites close to the infected stands. The application of this method may help plant protection services to detect new introductions of the pathogen within areas still free of chestnut blight and to prevent its establishment. It may also be useful for scientists involved in the epidemiology of the disease.

QPCR detection versus microscopy observations for assessing presence–absence of Didymosphenia geminata in Quebec rivers (Canada)

Microscopic versus qPCR (quantitative polymerase chain reaction) analyses were compared for the detection of Didymosphenia geminata in biofilms and water filtrates from seven Gaspésie rivers (Canada). For the qPCR approach, two DNA extraction kits (QIAamp DNA Micro Kit, Qiagen and PowerSoil DNA Isolation Kit, Mo Bio Laboratories) and two pairs of primers were considered. The pair of primers D602F/D753Rext did not amplify D. geminata DNA whereas the pair of primers D602F/D753R was specific for D. geminata. Presence-absence diagnosis based on qPCR and microscopic analyses were consistent: D. geminata was detected in six of the seven rivers, both in the biofilm and filtrate samples. However, technical replications were needed at certain sites to observe the presence of D. geminata cells by microscopy. This underscores the necessity of replicate analyses, which is cost-effective to achieve when using qPCR due to the capacity to process tens of samples in a single PCR run in the context of a large scale assessment.

Human T-lymphotropic virus types 1 and 2 are rare among intravenous drug users in Eastern Europe

BackgroundIn Europe, human T-lymphotropic virus (HTLV) type 2 mainly occurs among intravenous drug users (IDUs) with prevalence up to 15% and HTLV-1 among general population with prevalence <1%. However, there is no data regarding the prevalence of HTLV-1 or HTLV-2 in Eastern European IDUs population where HIV prevalence is relatively high. We aimed to determine the prevalence and genotypes of HTLV-1/HTLV-2 among IDUs and healthy volunteers in Estonia. MethodsThe study included 345 IDUs and 138 healthy volunteers. The presence of HTLV-1/HTLV-2 was determined by nested PCR; positive and negative controls were used in every PCR run. ResultsThe analysed IDUs resembled the IDUs of HIV epidemic in Estonia: mainly male (79%) with median age of 30years (interquartile range [IQR] 25–34), and prolonged duration of intravenous drug usage (11years; IQR 7–14). The prevalence exposure to blood-borne viral infections was high — 50% were HIV positive, 88% hepatitis C positive, 67% hepatitis B positive. Of IDUs, 64% reported receptive needle sharing in the past and 18% at least once a month during last six months. None of the IDUs carried HTLV-1 but there was a case of HTLV-2 (prevalence 0.3%; 95% CI 0.1–1.6). All healthy volunteers were HTLV-1 and HTLV-2 PCR negative. ConclusionThis is the first study investigating the prevalence of HTLV-1/HTLV-2 among high risk population and healthy volunteers in Eastern European region. Our results suggest that despite other widely spread blood-borne infections (e.g. HIV, HBV, HCV) HTLV-1 and HTLV-2 are rare among IDUs in Estonia.