PCR In Samples Research Articles

Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA

BackgroundNucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods.MethodsIn this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols.ResultsThe different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR.ConclusionsThe results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.

Diagnostic study for Toxoplasmosis by Serological test and PCR technique in Kirkuk province

Background: Severe Toxoplasma gondii infection in early pregnancy brings the risk of transferring the infection to the unborn baby with serious abnormality. The aim of identifying toxoplasmosis in early stage is to recognize mother-to-infant transmission for early cure to avoid undesirable complication. Using serological test to determine anti-Toxoplasma specific IgG and IgM antibodies possibly may not identify new or even previous infection. However, many molecular techniques are now being extensively used through the world among these methods polymerase chain reaction PCR technique emerged as the most widely accepted method for directly detection infectious agents. Thus, the goal in this study is to compare immunoassay by indirect (IgM and IgG) detection diagnostic technique and conventional PCR as molecular assay in diagnosis of T. gondii in whole blood of aborted females in Kirkuk province-Iraq.
 Methods: Here we applied indirect routine serological diagnosis test for detection toxoplasma specific IgM and IgG antibodies in females blood with spontaneous abortion and compare the efficiency of this method with PCR technique to detect the pathogenic protozoan T. gondii based on identification of (the B1 gene) as a target. Eighty-one females were incorporated in this study with a history of recurrent spontaneous abortions. All of these cases were limited to only females in the reproductive age (19-44 years). There are several serological diagnostic test kits available in Iraqi markets; however, the sensitivity and effectiveness of these commercial kits are not examined by most of the laboratories before they are obtained. Therefore, blood samples from female patients with single or repeated abortion were tested for detection specific anti-Toxoplasma antibodies using commercially available (Medical-Biozec) kit in Kirkuk province and compare that with the results are obtained from conventional PCR assay. 
 Results: The results showed that the conventional-PCR analysis is remarkably more sensitive than serological test, with a relatively high proportion of the positive samples for PCR compared with the serological diagnosis. The serological test results revealed that anti-T. gondii antibodies were detected in 14.81% of aborted women, 3.7% of them were positive for IgM, while 11.11% were positive for IgG. On the contrary, conventional PCR analysis found that 44.44% of aborted women showed positive result for B1 gene of T. gondii. Despite this, most of positive serology-aborted women exposed positive result for B1 gene of T. gondii, except two cases include false-positive IgM results

Simultaneous enumeration of Campylobacter jejuni and Salmonella enterica genome equivalents by melting curve analysis following duplex real time PCR in the presence of SYBR Green

Chicken meat and eggs contaminated with Salmonella enterica and Campylobacter jejuni are among the major causes of gastrointestinal infections in humans. Determining the numbers of these pathogens at various stages of the food supply chain is critical to the validation of steps designed to produce safer food. In the current study, duplex real time PCR in the presence of SYBR Green was carried out with DNA extracted from pure cultures of the two pathogens and from chicken meat samples spiked with them. The peak areas of derivative of dissociation curves (PADDC), obtained after 35 PCR cycles were calculated and plotted against known genome equivalents (GEs) in a standard curve. The method provided an estimation for the number of GEs in a 25 μL PCR sample when 102-105 GEs were present, similar to those obtained with duplex qPCR based on TaqMan probes by other authors, but with reduced costs.

The diversity of tick-borne bacteria and parasites in ticks collected from the Strandja Nature Park in south-eastern Bulgaria

BackgroundTicks are important carriers of many different zoonotic pathogens. To date, there are many studies about ticks and tick-borne pathogens (TBP), but only a few were carried out in Bulgaria. The present study intends to detect the prevalence of tick-borne bacteria and parasites occurring at the Black Sea in Bulgaria to evaluate the zoonotic potential of the tick-borne pathogens transmitted by ticks in this area.MethodsIn total, cDNA from 1541 ticks (Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp. and Rhipicephalus spp.) collected in Bulgaria by flagging method or from hosts was tested in pools of ten individuals each for Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi (s.l.), Rickettsia spp. and “Candidatus Neoehrlichia mikurensis” via conventional and quantitative real-time PCR. Subsequently, samples from positive pools were tested individually and a randomized selection of positive PCR samples was purified, sequenced, and analyzed.ResultsAltogether, 23.2% of ticks were infected with at least one of the tested pathogens. The highest infection levels were noted in nymphs (32.3%) and females (27.5%). Very high prevalence was detected for Rickettsia spp. (48.3%), followed by A. phagocytophilum (6.2%), Borrelia burgdorferi (s.l.) (1.7%), Babesia spp. (0.4%) and “Ca. Neoehrlichia mikurensis” (0.1%). Co-infections were found in 2.5% of the tested ticks (mainly Ixodes spp.). Sequencing revealed the presence of Rickettsia monacensis, R. helvetica, and R. aeschlimannii, Babesia microti and B. caballi, and Theileria buffeli and Borrelia afzelli.ConclusionThis study shows very high prevalence of zoonotic Rickettsia spp. in ticks from Bulgaria and moderate to low prevalence for all other pathogens tested. One should take into account that tick bites from this area could lead to Rickettsia infection in humans and mammals.

ANALISIS FILOGENETIK DENGUE DI INDONESIA

Molecular epidemiology is needed to solve the problem for endemic Dengue Hemorrhagic Fever in Indonesia.This research has been carried out consisting of 525 Dengue Hemorrhagic Fever sera, according to the WHO criteria.These sera were collected from 19 cities in Indonesia comprising the islands of Sumatera, Batam, Kalimantan, Sulawesi, Papua, Java,Bali and Lombok from 2003 until 2005. The immune response profile was as follows 57.14% (300/525) secondary infection, 12.57%(66/525) primary infection, 4.20% (22/525) equivocal and 26.09% (137/525) negative. From 192 PCR samples, 100 (52%) serawere positive, consisting of 65% DEN-2, 15% DEN-3, 12% DEN-4 and 8% DEN-1. Homology analysis showed nucleotide differences incapsid region DEN-2 serotypes, while DEN-3 serotypes were relatively consistent. Phylogenetic analysis using envelope (E) gene revealedthat the Cosmopolitan genotype from Gorontalo in 2005, is currently circulating locally, with the potential to cause a severe hemorrhagicdisease. Members of this genotype were closely related to viruses from Malaysia, Singapore, Thailand, Philippines and Australia. Theisolate from Jakarta, 2003 showed DEN-3 with I genotype. This genotype was similar to the isolate from Indonesia 1978, 1985, andalso from Thailand 1992, Philippines 1997, and Fiji 1992. These results showed Cosmopolitan genotype from DEN-2 was similar toSoutheast Asia countries. It was also revealed that genotype-I from DEN-3 showed no change over the years since 1978.

Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil

BackgroundThe hypotheses put forward to explain the malaria transmission cycle in extra-Amazonian Brazil, an area of very low malaria incidence, are based on either a zoonotic scenario involving simian malaria, or a scenario in which asymptomatic carriers play an important role.ObjectivesTo determine the incidence of asymptomatic infection by detecting Plasmodium spp. DNA and its role in residual malaria transmission in a non-Amazonian region of Brazil.MethodsUpon the report of the first malaria case in 2010 in the Atlantic Forest region of the state of Espírito Santo, inhabitants within a 2 km radius were invited to participate in a follow-up study. After providing signed informed consent forms, inhabitants filled out a questionnaire and gave blood samples for PCR, and thick and thin smears. Follow-up visits were performed every 3 months over a 21 month period, when new samples were collected and information was updated.ResultsNinety-two individuals were initially included for follow-up. At the first collection, all of them were clearly asymptomatic. One individual was positive for Plasmodium vivax, one for Plasmodium malariae and one for both P. vivax and P. malariae, corresponding to a prevalence of 3.4% (2.3% for each species). During follow-up, four new PCR-positive cases (two for each species) were recorded, corresponding to an incidence of 2.5 infections per 100 person-years or 1.25 infections per 100 person-years for each species. A mathematical transmission model was applied, using a low frequency of human carriers and the vector density in the region, and calculated based on previous studies in the same locality whose results were subjected to a linear regression. This analysis suggests that the transmission chain is unlikely to be based solely on human carriers, regardless of whether they are symptomatic or not.ConclusionThe low incidence of cases and the low frequency of asymptomatic malaria carriers investigated make it unlikely that the transmission chain in the region is based solely on human hosts, as cases are isolated one from another by hundreds of kilometers and frequently by long periods of time, reinforcing instead the hypothesis of zoonotic transmission.

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 101 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 105 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis.

Seek and Find! PCR analyses of skin infections in West-European travelers returning from abroad with an eschar.

Skin infections are among the leading causes of diseases in travelers. Diagnosing pathogens could be difficult. We applied molecular assays for the diagnostic of a large collection of skin biopsies and swabs from travelers with suspected skin infections. All samples were tested by qPCR for Coxiella burnetti, Bartonella sp., Rickettsia sp., Borrelia sp., Ehrlichia sp., Tropheryma whipplei, Francisella tularensis, Mycobacteria sp., Staphylococcus aureus, Streptococcus pyogenes, Leishmania spp., Ortho poxvirus and Para poxvirus and then screened for the presence of bacteria by PCR amplification and sequencing, targeting the 16S rRNA gene. From January 2009 to January 2017, 100 international travelers presenting with a suspected skin infection were enrolled. We detected 51 patients with an identified pathogen on skin samples. Travelers presenting with eschars were more likely to have a positive PCR sample (n = 44/76, 57.9%) compared to other patients (n = 7/24, 29.2%). Spotted fever group Rickettsia (n = 28) was the most frequently detected pathogens (19 R. africae, 6 R. conorii, 3 R. mongolitimonae); S. aureus were detected in 11 patients; S. pyogenes in 3; Leishmania sp.; M. leprae and B. henselae in 1 patient, respectively. By targeting the most commonly encountered causative agents of travel-related skin infections, our strategy provides a sensitive and rapid diagnostic method.

Optimal Reference Gene Selection for Expression Studies in Human Reticulocytes

Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports. Real-time quantitative PCR was performed on reticulocytes in 20 healthy volunteers, 15 HS patients, and 10 βTI patients. Threshold cycle values were compared with fold-change method and RefFinder software. The stable reference genes recommended by RefFinder were validated with SLC4A1 and flow cytometric eosin-5'-maleimide binding assay values in HS patients and HBG2 and high performance liquid chromatography-derived percentage of hemoglobin F in βTI. Comprehensive ranking predicted MPP1 and GAPDH as optimal reference genes for reticulocytes that were not affected in HS and βTI. This was further confirmed on validation with eosin-5'-maleimide results and percentage of hemoglobin F in HS and βTI patients, respectively. Hence, MPP1 and GAPDH are good reference genes for reticulocyte expression studies compared with ACTB and RN18S1, the two most commonly used reference genes.

Abstract P2-09-04: Association of intrinsic subtype and immune genes with pathological complete response in the OPTIHER-HEART phase II clinical trial following neoadjuvant trastuzumab/pertuzumab-based chemotherapy in HER2-positive breast cancer

Abstract Background. In HER2-positive early breast cancer, neoadjuvant therapy based on dual HER2 blockade with trastuzumab and pertuzumab in combination with chemotherapy is associated with high rates of pCR. The OPTIHER-HEART trial examined the safety of the neoadjuvant dual blockade added to paclitaxel and liposomal doxorubicin (Gávila et al, SABCS 2016). In this analysis, we examined the intrinsic subtypes and immune genes in baseline and surgical samples from the OPTIHER-HEART trial. Methods.OPTIHER-HEART was a multicenter single-arm phase II study of 18 weeks of neoadjuvant pertuzumab, weekly trastuzumab, liposomal doxorubicin (50 mg/m2 every 21 days) and weekly paclitaxel 80 mg/m2 in patients with stage II-IIIB HER2-positive breast cancer. pCR was defined as absence of infiltrating tumor in the breast and axilla. The expression of 55 breast cancer-related and immune genes in baseline samples and surgical specimens was measured using the nCounter platform. Intrinsic subtypes were determined by the PAM50 gene expression predictor. Univariate and multivariable logistic regression models adjusted for tumor size, ER status, age and nodal status was performed. Results. A total of 58 of the 83 (69.8%) baseline tumor samples were available. 51.7% (n=30) were classified as HER2 enriched (HER2-E) followed by Normal-like (15.5%; n=9), Basal-like (12.0%; n=7), Luminal A (10.3%; n=6), and Luminal B (10.3%; n=6). HER2-E was the most frequent subtype in both hormone receptor (HR)-negative (50.0%) and HR-positive (52.0%) tumors. The pCR rates varied according to intrinsic subtype (p<0.001). The highest rate of pCR was observed in Basal-like (85.7%) and HER2-E (83.3%), followed by Normal-like (44.4%), Luminal A (33.3%) and Luminal B (16.6%). HER2-E tumors were associated with higher pCR rates compared to non-HER2-E tumors (83.3% vs 46.5%, unadjusted odds ratio = 5.76, 95% CI 1.71 – 19.42, P=0.004), even after adjusting for HR status, size, age and lymph node involvement (odds ratio = 13.50, 95% CI2.51 – 72.52, P=0.002). Among the 55 genes, 14 (25.5%) were statistically significantly associated with pCR in univariate analysis (false discovery rate < 1%). Genes associated with pCR were 17q12-21 amplicon genes (e.g. GRB7 and ERBB2) and immune genes (e.g. CD8A and PD1). Genes associated with residual disease were luminal-related genes (e.g. ESR1 and PGR). Among the 14 genes, only 7 (i.e. CDH3, CD8A, PD1, EGFR, ESR1, SLC39A6, NAT1) were significantly associated with pCR beyond intrinsic subtype (HER2-E vs non-HER2-E). Finally, a total of 58 of the 80 (72.5%) surgical specimens were profiled. 77% of the samples were classified as Normal-like, followed by Luminal A (20%), Luminal B (2%) and HER2-E (2%). Normal-like was identified in a higher proportion in pCR samples (90%) compared to samples with residual disease (47%) (p<0.001). Conclusion. HER2-E subtype and immune genes are independently associated with pCR after dual HER2 blockade and multi-agent chemotherapy. Subtyping of surgical specimens might provide additional response and outcome data and warrants further evaluation. Citation Format: Gavila J, Oliveira M, Pascual T, Pérez J, Canes J, González X, Paré L, Calvo I, Ciruelos E, Muñoz M, Virizuela JA, Ruiz I, Andrés R, Perelló A, Martínez J, Morales S, Marín M, Martínez D, Quero JC, Llombart-Cussac A, Prat A. Association of intrinsic subtype and immune genes with pathological complete response in the OPTIHER-HEART phase II clinical trial following neoadjuvant trastuzumab/pertuzumab-based chemotherapy in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-04.

Comparison of Dengue viral nonstructural protein 1 antigen testing kits

Objective: To investigate the sensitivity and specificity of commercial nonstructural protein 1 (NS1) testing kits for Dengue fever diagnose, and provide the evidence for diagnostic criteria revision. Methods: 300 PCR or virus isolation positive blood samples for dengue virus were collected from sentinel hospitals for dengue surveillance in Guangzhou, Dongguang and Zhongshang from May 2015 to Nov. 2016. At the same time, 308 PCR negative samples for Dengue virus were collected as control group. The information of the sample was collected using questionnaires. These samples were tested using imported and domestic ELISA and the colloidal gold-labeled kits that were widely used for detecting dengue NS1. Sensitivity, specificity and coincidence were calculated and analyzed, and Z hongshan's result was regarded as the reslut of the third part. Results: The positive group includes 133 males and 167 females, average ages are 47.2±13.3, 179, 110 and 11 of them is Dengue Ⅰ, Ⅱ and Ⅲ respectively. The negative group includes 154 males and 154 females, average ages are (40.1±11.6) years old. The sensitivity of domestic ELISA Kits (94.5%) is less than imported (99.5%), and the result has statistical significance (χ(2)=8.59, P=0.030), the specificity is 99.7% and 97.7% respectively; The sensitivity of imported and domestic the colloidal gold-labeled Kits is 97.5% and 96.5% respectively, both of specificities are 100%. The sensitivity and specificity of Dengue Ⅰ for NS1 test are more than 97.0%. The sensitivity of domestic ELISA and gold-labeled Kits is 90.0% and 95.0%, and the specificity is 96.8% and 100% respectively for Dengue Ⅱ test. The sensitivity of imported ELISA and gold-labeled Kits is 100% and 98.0%, and the specificity is 99.4% and 100% respectively for Dengue Ⅱ test. The result of the third party show the sensitivity and specificity of domestic ELISA and gold-labeled Kits are 90.0% and 98.0%, the differences has statistical significance (χ(2)=5.67, P=0.020). Conclusion: NS1 testing can be used as early dengue fever diagnose for higher sensitivity and specificity.

Abstract LB-B22: TNBC kinase prioritization for targeted therapy after chemotherapy resistance

Abstract Introduction: Triple negative breast cancer accounts for 10-15% of all breast cancers yet contribute to high recurrence and metastasis that leads to the death of patients. Lack of molecular therapeutic target for TNBC leaves the chemotherapy as only available options while many develop resistance to chemotherapeutics, leaving patients with TNBC at risk of accumulative toxicities and death. Thus, it is critical to identify unique molecular kinases that contribute to chemotherapy resistance. We hypothesize that differential level of kinase expression stratified based on the chemotherapy response, to discover putative targets. Methods and Material: We performed an in silico search of kinases using mRNA microarray data that are expressed with higher proportion in TNBC patient samples based on the chemotherapeutic response, to identify kinases that contribute to the resistance to neoadjuvant chemotherapy using mRNA sequencing data of 132 patients with TNBC. We then prioritize the putative resistance related kinases, based on priority scoring system we have developed, to select the top candidate kinase for further therapeutics development. Result: Eleven kinases were differentially expressed between the pCR and non-pCR patients samples at baseline, and prioritized based on our scoring system. MELK was selected as a top target kinase given the highest score and biological validation, and currently available inhibitor. Pre-clinical studies of MELK has shown efficacy in vitro and in vivo models. Conclusion: First in human trial of oral MELK inhibitor is currently launched to be tested in actual clinical setting in patients with TNBC, that will offer the opportunity for us to validate our prioritization scoring system and pre-clinical validation. Further clinical validation, which is ongoing, will support our in silico screening followed by a prioritization to select kinase inhibitor development in chemotherapy resistant TNBC. Citation Format: Bora Lim, In Ah Park, Ying Wang, Lakshmi Bollu, Powel Brown, Keith Baggerly, Funda Meric-Bernstam, Naoto Ueno. TNBC kinase prioritization for targeted therapy after chemotherapy resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B22.

Chlamydia psittaci and C. avium in feral pigeon (Columba livia domestica) droppings in two cities in the Netherlands

Background: Feral pigeons (Columba livia domestica) live and breed in many city centres and contact with their droppings can be a hazard for human health if the birds carry Chlamydia psittaci.Objective: The aim of this study was to establish whether pigeon droppings in two Dutch cities (Utrecht and Haarlem) contain C. psittaci and/or C. avium, which could be a potential hazard for transmission to humans.Methods: In May 2017 seven feral pigeon ‘hot spots’ with between 5 and 40+ pigeons present were identified in two cities by visual observations over two days. During the following ten days fresh droppings were collected at these hot spots and the samples were pooled per three droppings to achieve 40–41 samples per city. Samples were analysed for Chlamydia DNA with a broad range 23S Chlamydiaceae Real-Time PCR and positive samples were tested with a specific C. psittaci and C. avium Real-Time PCR. Positive C. psittaci samples were genotyped.Results: C. psittaci and C. avium were detected in both cities. For C. psittaci the prevalences in Utrecht and Haarlem were 2.4% and 7.5%, respectively; for C. avium 36.6% and 20.0%, respectively. One sample contained both species. All C. psittaci samples belonged to genotype B.Conclusion: C. psittaci and C. avium are present in feral pigeon droppings in Utrecht and Haarlem. Human contact with droppings from infected pigeons or inhalation of dust from dried droppings represent a potential hazard to public health.

Phylogenetic Characterization of Infectious Bursal Disease (IBD) Viruses Isolated From Field Outbreaks in Chickens From Behera And Alexandria Governorates, Egypt

Infectious bursal disease continue to be happened in Egypt causing a great economic losses in chickens. Thirty chicken flocks showing clinical signs suspected to be due to IBDV infection have been investigated from Behera and Alexandria governorates. Twelve flocks are confirmed to be infected with IBDV by RT/PCR showing a band of 620 bp on agar gel using a specific primer of VP2 gene of IBDV. Eight positive PCR samples were chosen for further characterization by sequencing analysis. Seven isolates were characterized as vvIBDV and one as classical IBDV. The molecular characterization of isolated IBD field strains in the present study indicated a continuous circulation of vvIBDV although of intensive vaccination programs.

Electrochemical Detection of Bacillus anthracis Protective Antigen Gene Using DNA Biosensor Based on Stem−Loop Probe

The presented study is focused on the development of repetitive, fast and versatile “on-off” signaling electrochemical genosensor for detection of pagA gene of Bacillus anthracis. Such a sensor will be further used in the portable devices dedicated to molecular diagnostic. The DNA molecular beacon probe was immobilized onto gold electrodes in its folded state through the alkanethiol linker at the 5’-end, while the 3’-end was labeled with methylene blue redox molecule. Initial optimization was conducted for HPLC purified cDNA. Nonetheless application of such prepared biosensor for unpurified PCR product required further investigations. As the sensor response was significantly dependent on the stem-loop architecture, the three different hairpin probes were analyzed. The detected sequence was interior to amplicon, identical as in case of cDNA. The developed “on-off” genosensor allows for detection, in 5 minute, the pagA gene sequence at the level of 5.7 nM with 22.9–86.0 nM linear detection range also in the great excess of other DNA presented in the sample. Both LOD and linear range were significantly improved in case of longer assay time. The biosensor proved its applicability also in detection of pagA gene of Bacillus anthracis in PCR samples amplified with reduced number of cycles.

Selection of internal references for qRT-PCR assays of human hepatocellular carcinoma cell lines.

Selecting internal references is important for normalizing the loading quantity of samples in quantitative reverse-transcription PCR (qRT-PCR). In the present study, a systematic evaluation of reference genes among nine hepatocellular carcinoma (HCC) cell lines was conducted. After screening the microarray assay data of ten HCC cell lines, 19 candidate reference genes were preselected and then evaluated by qRT-PCR, together with ACTB, GAPDH, HPRT1 and TUBB. The expression evenness of these candidate genes was evaluated using RefFinder. The stabilities of the reference genes were further evaluated under different experimental perturbations in Huh-7 and MHCC-97L, and the applicability of the reference genes was assessed by measuring the mRNA expression of CCND1, CCND3, CDK4 and CDK6 under sorafenib treatment in Huh-7. Results showed that TFG and SFRS4 are among the most reliable reference genes, and ACTB ranks third and acts quite well as a classical choice, whereas GAPDH, HPRT1 and TUBB are not proper reference genes in qRT-PCR assays among the HCC cell lines. SFRS4, YWHAB, SFRS4 and CNPY3 are the most stable reference genes of the MHCC-97L under the perturbations of chemotherapy, oxidative stress, starvation and hypoxia respectively, whereas YWHAB is the most stable one of Huh-7 under all perturbations. GAPDH is recommended as a reference gene under chemotherapy perturbations. YWHAB and UBE2B, TMED2 and TSFM, and GAPDH and TSFM are the two best reference genes under oxidative stress, starvation and hypoxia perturbations respectively. TSFM is stable in both cell lines across all the perturbations.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Influenza virus infections from 0 to 2 years of age: A birth cohort study.

Influenza vaccine has been recommended in Finland since 2007 for all children of 6-35 months of age and in 2009 for those ≥6 months against pandemic influenza. We investigated the incidence of influenza and vaccine effectiveness in a birth cohort of children in 2008-2011. We followed 923 children from birth to 2 years of age for respiratory tract infections. A nasal swab sample for PCR for influenza A and B viruses was taken at the onset of acute respiratory infections. Samples were collected either at the study clinic or at home by parents. Vaccination data was retrieved from the health registries. Vaccination coverage of children aged 6-23 months was 22-47% against seasonal influenza and 80% against the A(H1N1)pdm09 virus in the pandemic season 2009-2010. During 3 influenza seasons, 1607 nasal swab samples were collected. Influenza was confirmed in 56 (6.1%) of 923 children (16 A(H1N1), 14 A(H3N2), and 26B viruses). The incidence of influenza was 5.1% in 2008-2009, 2.7% in 2009-2010, and 5.0% in 2010-2011. Effectiveness of the adjuvanted vaccine against the pandemic influenza A(H1N1)pdm09 was 97% (95% confidence interval, 76-100%). Three children with influenza were hospitalized. The yearly incidence of seasonal influenza was 5% in this cohort of very young children with variable influenza vaccine coverage. Adjuvanted vaccine against the pandemic influenza was highly effective. Both seasonal and pandemic influenza cases were mostly non-severe.

Impact of HPV vaccination with Gardasil\xae in Switzerland

BackgroundGardasil®, a quadrivalent vaccine targeting low-risk (6, 11) and high-risk (16, 18) human papillomaviruses (HPV), has been offered to 11–14 year-old schoolgirls in Switzerland since 2008. To evaluate its success and its potential impact on cervical cancer screening, HPV genotypes were examined in 18-year-old girls five years later (sub-study 1) and in outpatients participating to cervical cancer screening before and after vaccine implementation (sub-study 2).MethodsFor sub-study 1, 3726 females aged 18 in 2013 were invited to fill a questionnaire on personal demographics and HPV risk factors and to provide a self-collected cervicovaginal sample for HPV genotyping and Chlamydia trachomatis PCR. Personal data were evaluated by univariable and multivariable statistics. In sub-study 2, the proportion of the vaccine-type HPV among anogenital HPV was examined with archived genotyping data of 8039 outpatients participating to cervical cancer screening from 1999 till 2015. The yearly evolution of this proportion was evaluated by segmented logistic regression.Results690 (18.5%) women participated to sub-study 1 and 327 (8.8%) provided a self-collected sample. Prevalence of Chlamydia trachomatis (4.6%) and demographics confirmed that the subjects were representative of sexually-active Swiss young women. Vaccine (five-year coverage: 77.5%) was preferentially accepted by contraceptive-pill users (P = 0.001) and samples were mainly provided by sexually-active subjects (P < 0.001). The proportion (4%) of the vaccine-type HPV in this population was lower than in sub-study 2 outpatients (n = 849, <26 years old) in the pre-vaccine era (25.7%). The proportion of the high-risk vaccine-type HPV decreased significantly (59%, P = 0.0048) in the outpatients during the post-vaccine era, yet this decrease was restricted to those aged less than 26 years (n = 673, P < 0.0001).ConclusionsThe low proportion of vaccine-type HPV in 18-year-old females and its rapid decrease in young women participating to cervical cancer screening extend the success of HPV vaccination to Switzerland. Our data suggest that cervical cancer screening is now entering a stage of reduced proportion of HPV16 and/or 18 in samples reported positive by cytology. In view of the high likelihood of reduced clinical specificity of cytology, primary screening modalities involving HPV testing and cytology should now be re-evaluated in Switzerland.

Electrochemical DNA biosensor based on gold nanoparticles and partially reduced graphene oxide modified electrode for the detection of Listeria monocytogenes hly gene sequence

In this study an electrochemical DNA biosensor with gold nanoparticles (AuNPs) and partially reduced graphene oxide (p-RGO) modified electrode was constructed to detect Listeria monocytogenes hly gene sequence. AuNPs were electrodeposited on the surface of carbon ionic liquid electrode (CILE) and then p-RGO was further deposited at controlled electroreduction conditions. On the p-RGO surface unreduced oxygenal groups were present and could covalently interact with the amine group at the 5′-end of probe ssDNA through carbodiimide linkage, and the resulted ssDNA/p-RGO/AuNPs/CILE could be used to hybridize with the target ssDNA sequence. Differential pulse voltammetry was applied to monitor the DNA hybridization with methylene blue as electrochemical indicator. Under the optimal conditions, the electrochemical biosensor could be applied to the detection of target DNA sequence in the concentration range from 1.0×10−13–1.0×10−6mol/L with the detection limit of 3.17×10−14mol/L (3S0/S). The sensor exhibited good stability and selectivity to one or three-base mismatched ssDNA sequences. PCR sample of Listeria monocytogenes hly gene sequence was successfully detected, indicating the real application of this biosensor.