TaqMan Probe-based Real-time PCR Research Articles

Characterization and Performance Evaluation of Anaerobic Night Soil Biodegrading Inoculum

The efficiency of anaerobic night soil (human excreta) biodegradation depends on the characteristics of the inoculum used. This anaerobic microbial inoculum (AMI) contains a microbial consortium, including rate-limiting methanogens whose growth depends on various physicochemical and biological parameters necessitating the inoculum characterisation essential for optimum night soil (NS) biodegradation. The inoculum contained 24.567 g/L (±0.07) total solid, 5.022 (±0.57) g/L total dissolved solid, 18.148 (±0.0.058) g/L volatile solid (VS), and 7.0±0.2 pH. The Gas chromatography analysis confirmed the presence of a small amount of volatile fatty acid (VFA), (≤ 4 mM except propionate) showing rapid conversion of VFA to methane, and 55% methane. Further, the presence of methanosaetaceae group of methanogens was detected through Taqman probe-based real-time PCR. The inoculum performance was evaluated for NS biodegradation in semicontinuous mode of feeding in four 2 L anaerobic biodigesters (2D HRT, 4D HRT, 5D HRT, and 10D HRT) with varying hydraulic retention time (HRT) (2, 5, 8, and 10 days, respectively) at mesophilic temperature (35°C). After 5 HRT, VS reduction for 2D HRT and 5D HRT was <40% while for 8D HRT and 10D HRT was >45-50% and >45-60%, respectively. The methane content was <20% in biogas for 2 and 5 days HRT while for 8 and 10 days >45-50% and 50-65% methane, respectively. The % VS reduction and methane content showed the overfeeding of NS in biodigesters 2D HRT and 5D HRT while 8D HRT showed towards optimum and 10D HRT optimum with improved VS reduction and methane content.

Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application.

Diagnostic utility of intranodular saline lavage in TaqMan probe-based real-time PCR diagnosis of lumpy skin disease

Lumpy skin disease (LSD), caused by the poxvirus lumpy skin disease virus has entered India, causing serious economic consequences for the Indian dairy sector. To evaluate the diagnostic value of intranodular saline lavage in the identification of LSD-infected animals, skin nodule tissue biopsy, intranodular saline lavage, whole blood, and nasal swab specimens were collected simultaneously from LSD-symptomatic animals. TaqMan probe-based real-time PCR was performed targeting the EEV glycoprotein gene (LSDV126) and the results were compared. qPCR analysis could detect LSDV in all the skin nodule tissue biopsy and intranodular saline lavage. The mean cycle threshold (Cq) values of skin nodule tissue biopsy samples (23.94 ± 2.97) were also comparable with intranodular saline lavage (25.23 ± 2.41) samples. Whole blood and nasal swabs have an extremely poor detection rate for PCR-based viral detection.Our findings demonstrate that intranodular saline lavage is an excellent alternative specimen for the detection of LSDV. Taking into consideration, the simplicity of specimen collection, intranodular saline lavage could be adopted in field conditions for accurate LSDV surveillance testing.

A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/μL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/μL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

TaqMan probe-based commercial real-time (RT) PCR kits are expensive but most frequently used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections needs to meet the challenge of testing more persons at a reasonable cost. This study developed a simple and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay targeting two virus-specific genes along with a host-specific internal control. A total of 180 randomly selected samples portioning into two subsets based on crude and high-quality RNA extraction were used to compare this assay with a nationwide available commercial kit (Sansure Biotech Inc., (Hunan, China)), so that we could analyze the variation and validity of this in-house developed method. Our customized-designed primers can specifically detect the viral RNA likewise Sansure. We separately optimized SYBR Green RT-PCR reaction of N, E, S, and RdRp genes based on singleplex melting curve analysis at the initial stage. After several rounds of optimization on multiplex assays of different primer combinations, the optimized method finally targeted N and E genes of the SARS-CoV-2 virus, together with the β-actin gene of the host as an internal control. Comparing with the Sansure commercial kit, our proposed assay provided up to 97% specificity and 93% sensitivity. The cost of each sample processing ranged between ~2 and ~6 USD depending on the purification level of extracted RNA template. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in low-income countries.

DETECTION OF SPLENDIDOFILARIA SP. (ONCHOCERCIDAE:SPLENDIDOFILARIINAE) MICROFILARIA WITHIN ALASKAN GROUND-DWELLING BIRDS IN THE GROUSE SUBFAMILY TETRAONINAE USING TAQMAN PROBE-BASED REAL-TIME PCR.

Grouse and ptarmigan (Galliformes) harbor fairly diverse helminth faunas that can impact the host's health, including filarial nematodes in the genus Splendidofilaria. As host and parasite distributions are predicted to shift in response to recent climate change, novel parasites may be introduced into a region and impose additional stressors on bird populations. Limited information is available on the prevalence of filariasis in Alaska galliforms. To date, no molecular surveys have been completed. Past studies relied on examining blood smears or total body necropsies, which are time-consuming and may not detect filarial parasites with low prevalence in hosts. Therefore, we developed a TaqMan probe-based real-time PCR assay targeting the cytochrome c oxidase 1 gene (COI) of Splendidofilaria to decrease processing times and increase sensitivity as well as provide baseline data on the diversity of filariid infections in galliform species in Alaska. We screened a combined total of 708 galliform samples (678 unique individual birds) from different tissues (blood, muscle, and lung) for the presence of filarial DNA across the state of Alaska. Real-time PCR screening revealed an overall prevalence of filarial infection of 9.5% across species: Bonasa umbellus (0%, n = 23), Dendragapus fuliginosus (0%, n = 8), Falcipennis canadensis (26.8%, n = 198), Lagopus lagopus (2.6%, n = 274), Lagopus leucura (0%, n = 23), Lagopus muta (3%, n = 166), and Tympanuchus phasianellus (12.5%, n = 16). We observed microfilarial infections throughout most of Alaska except in Arctic regions and the Aleutian Islands where viable vectors may not be present.

Potential impact of TNFAIP3 rs6920220 and DEFB1 rs1800972 gene polymorphisms on vitiligo in Egyptian patients

BackgroundVitiligo is a complicated disorder identified by advanced degeneration and loss of melanocytes. Some of the main factors that cause vitiligo are cytotoxicity, autoimmunity, along with several genetic factors. AimThe current study aims at evaluating the association of TNFAIP3 rs6920220 and DEFB1 rs1800972 gene polymorphisms as risk factors of non-segmental vitiligo in Egyptian patients. Patients and methodsThis study was conducted on 125 patients with non-segmental vitiligo and 110 age and gender-matched healthy controls. Genotyping of TNFAIP3 rs6920220 and DEFB1 rs1800972 polymorphisms were analyzed by TaqMan probe-based real-time PCR. ResultsSignificant differences in the genotypes and alleles distributions of both polymorphisms were detected between patients and controls. The AA and GA genotypes of TNFAIP3 rs6920220 increase the risk of vitiligo with OR 4.422 and 1.863 respectively. The (GA + AA) model reported risk with OR 2.016 compared to the GG genotype. The CG, GG genotypes compared to the CC genotype of DEFB1 rs1800972 were found to have an increased risk of vitiligo with OR1.7 and 2.865 respectively. The (GG+ CG) model had OR 1.856. The AA genotype, the A allele of TNFAIP3 rs6920220, the GG genotype, and the G allele of DEFB1 rs1800972 were more frequent in patients with the progressive course and those with a positive family history of other autoimmune diseases as compared to controls. ConclusionTNFAIP3 rs6920220 and DEFB1 rs1800972 polymorphisms could participate in the pathogenesis of vitiligo and might be considered as potential risk factors for vitiligo.

A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control

Lumpy skin disease (LSD) is a transboundary disease of economic importance affecting cattle and buffaloes. In South-Eastern Europe, immunization of cattle with homologous live attenuated vaccines for LSD control has prevented outbreaks since 2017, but has been associated with adverse reactions resembling disease symptoms. Thus, a diagnostic method suitable for disease surveillance in farms during vaccination campaigns with Neethling (Onderstepoort) and SIS type (Lumpyvax) live attenuated LSDV vaccines in Europe should be able to detect the wild type (WT) LSDV in animals with adverse reactions to the vaccines and samples with potentially high titers of the vaccine LSDV. To this end, a real-time PCR method targeting the EEV gene of LSDV was developed for the specific detection of WT strains, along with the use of beta-actin gene as an internal amplification control (IAC). Amplification efficiency of the WT virus target was 99.0% and 98.6%, in the presence and in the absence of high loads of vaccine LSDV, respectively. In the presence of 105.6 vaccine LSDV DNA copies, the limit of detection for WT LSDV was 12.6 DNA copies per reaction. The inter-assay CV was 0.04% for WT LSDV and 0.13% for beta-actin. The method can confirm diagnosis in suspect cases irrespective of the presence of the vaccine LSDV DNA by overcoming the masking effect of the WT LSDV. The simultaneous amplification of the beta-actin gene further assures the quality of diagnostic testing. The new method is a surveillance tool, complementing the DIVA real-time PCR during vaccination campaigns and can provide rapid insight on the targeted EEV gene in countries with novel and recombinant LSDV strains.

Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/μL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Genome-Wide Expression Difference of MicroRNAs in Basal Cell Carcinoma.

Distinct expression of the miRNAs has rarely been explored in basal cell carcinoma (BCC) of skin, and the regulatory role of miRNAs in BCC development remains quite opaque. Here, we collected control tissues from adjacent noncancerous skin (n = 15; control group) and tissues at tumor centers from patients with cheek BCC (n = 15; BCC group) using punch biopsies. After six small RNA sequencing- (sRNA-seq-) based miRNA expression profiles were generated for both BCC and controls, including three biological replicates, we conducted comparative analysis on the sRNA-seq dataset, discovering 181 differentially expressed miRNAs (DEMs) out of the 1,873 miRNAs in BCCs. In order to validate the sRNA-seq data, expression of 15 randomly selected DEMs was measured using the TaqMan probe-based quantitative real-time PCR. Functional analysis of predicted target genes of DEMs in BCCs shows that these miRNAs are primarily involved in various types of cancers, immune response, epithelial growth, and morphogenesis, as well as energy production and metabolism, indicating that BCC development is caused, at least in part, by changes in miRNA regulation for biological and disease processes. In particular, the “basal cell carcinoma pathways” were found to be enriched by predicted DEM targets, and regulatory relationships between DEMs and their targeted genes in this pathway were further uncovered. These results revealed the association between BCCs and abundant miRNA molecules that regulate target genes, functional modules, and signaling pathways in carcinogenesis.

Molecular detection and clinical characteristics of bacterial and viral main etiological agents causing respiratory tract infections in Tehran, Iran

BackgroundRespiratory tract infections are one of the most important infections among military personals, worldwide. The present study aimed to survey the prevalence of bacterial and viral main etiological agents causing respiratory tract infections among military personnel in Iran. MethodsA cross-sectional study was performed from November 2020 to March 2021. Nasopharyngeal swabs were taken from patients, military personnel with respiratory tract infection symptoms. Detection of coronavirus disease 2019 (COVID-19) was performed with one step qRT-PCR method. TaqMan probe-based real-time PCR assay was used for the detection of influenza A and B viruses. The prevalence of adenovirus and Mycoplasma pneumoniae was determined using nested PCR. Moreover, Bordetella pertussis and Streptococcus pyogenes were identified by conventional PCR assay. The detection of Haemophilus influenzae was performed by the multiplex PCR method. ResultsOverall, 145 patients were included. Among viral pathogens, COVID-19, influenza A virus, and adenovirus were identified in 85.5%, 4.1%, and 1.4% of patients, respectively. Influenza B virus was not detected among military personnel. The frequency of bacteria etiological agents was as follows: S. pyogenes (2%), M. pneumoniae (0.7%), H. influenzae (0%), and B. pertussis (0%). Muscle aches (75.9%), headache (70.3%), lethargy (69%), cough (66.2%), stuffy nose (56.6%), fever (53.8%), and sore throat (53.1%) were among the most common clinical symptoms. ConclusionsResults showed that the military personals are the susceptible group to COVID-19 infection. Therefore, the accurate detection and implementation of control strategies such as vaccination are necessary.

Molecular Detection Assays for Rapid Field-Detection of Rice Sheath Blight.

Rhizoctonia solani (Rs), a soil-borne fungal pathogen, can result in rice sheath blight (ShB), which causes yield loss. To prevent outbreaks of ShB and enhance the sustainability of rice production, it is critical to develop a rapid ShB detection method for specific, fast, and on-site disease management. In this study, a reagent for the rapid extraction of this pathogen was developed for on-site detection. The specificity and sensitivity of a novel SMS RS1-F/SMS RS1-R primer set and a ITS1/GMRS-3 reference primer set were tested, while four different extraction protocols for ShB were developed. Moreover, intraday and interday assays were performed to evaluate the reproducibility of the detection methods developed. The results indicated that all of the developed protocols are suitable for use in detecting ShB. In addition, all the samples of infected rice yielded positive Rs detection results when subjected to TaqMan probe-based real-time PCR and SYBR green-based real-time PCR (SMS RS1-F/SMS RS1-R) tests in which automatic magnetic bead-based DNA extraction was performed. These results indicated that the two molecular detection protocols were suitable for the field diagnosis of ShB for all asymptomatic and symptomatic rice samples.

Effects of clopidogrel resistence and CYP2C19 genotype on the clinical prognosis of patients with acute coronary syndrome undergoing percutaneous coronary intervention

Objective: To investigate the effects of clopidogrel resistence and CYP2C19 genotype on the clinical prognosis of acute coronary syndrome(ACS) patients undergoing percutaneous coronary intervention(PCI). Methods: This study was a retrospective cohort study. ACS patients who underwent PCI in Beijing Anzhen Hospital from October 2015 to January 2017 were recruited. The inhibition rate of adenosine diphosphate(ADP) was monitored by thromboelastography. All of these patients were divided into clopidogrel resistance and non-resistance group according to the monitoring results. CYP2C19 genotype was detected by TaqMan probe-based real-time quantitative PCR. Patients were divided into slow, medium and fast metabolic group, according to the CYP2C19 genotype. After 12 months of follow-up, the end points included all-cause death, cardiac death, angina, myocardial infarction, stent thrombosis, ischemic stroke and hemorrhage were collected. Combined thrombotic events were defined as a composite of angina, myocardial infarction, stent thrombosis and ischemic stroke. The differences of the incidence of clinical events between groups were compared. Cox regression was used to analyze the effects of clopidogrel resistance and CYP2C19 genotype on the combined thrombotic events, cardiac death and hemorrhage. Results: A total of 1 696 patients were included, and the age was (59.4±9.6) years, with 1 280(75.5%) males. There were 471 cases(27.8%) in clopidogrel resistance group, and 1 225 cases(72.2%) in clopidogrel non-resistance group. There were 218 patients(12.9%) were in slow metabolic group, 668(39.4%) in medium metabolic group, and 810 (47.8%) in fast metabolic group. The median follow-up time was 13.3 months, and 131 cases were lost to follow-up, with a loss follow-up rate of 7.7%. Compared with the clopidogrel non-resistance group, the clopidogrel resistance group had a higher incidence of myocardial infarction(7.6%(36/471) vs. 5.1%(62/1 225), P=0.041), a lower incidence of hemorrhage (13.2%(62/471) vs. 17.9%(219/1 225), P=0.020) and minor hemorrhage(11.5%(54/471) vs. 15.8% (194/1 225), P=0.022). There were no statistically significant difference in all-cause death, cardiac death, angina, stent thrombosis, ischemic stroke and severe bleeding between clopidogrel resistance and non-resistance group(all P>0.05). There was no statistically significant difference in the incidence of endpoint events among different CYP2C19 genotypes (all P>0.05). Cox regression analysis showed that clopidogrel resistance was an independent factor of combined thrombotic events (OR=2.334, 95%CI 1.215-4.443, P=0.016) and bleeding events (OR=0.481, 95%CI 0.174-0.901, P=0.023). While CYP2C19 genotype was not independent factor for combined thrombotic events, cardiac death and hemorrhage (all P>0.05). Conclusion: For ACS patients after PCI, clopidogrel resistance can increase the risk of combined thrombotic events, but also reduce the risk of bleeding; while CYP2C19 genotype is not an independent factor for clinical prognosis.

Molecular epidemiological surveillance of Africa and Asia imported malaria in Wuhan, Central China: comparison of diagnostic tools during 2011\u20132018

BackgroundMalaria remains a serious public health problem globally. As the elimination of indigenous malaria continues in China, imported malaria has gradually become a major health hazard. Well-timed and accurate diagnoses could support the timely implementation of therapeutic schedules, reveal the prevalence of imported malaria and avoid transmission of the disease.MethodsBlood samples were collected in Wuhan, China, from August 2011 to December 2018. All patients accepted microscopy and rapid diagnosis test (RDT) examinations. Subsequently, each of the positive or suspected positive cases was tested for four human-infectious Plasmodium species by using 18S rRNA-based nested PCR and Taqman probe-based real-time PCR. The results of the microscopy and the two molecular diagnostic methods were analysed. Importation origins were traced by country, and the prevalence of Plasmodium species was analysed by year.ResultsA total of 296 blood samples, including 288 that were microscopy and RDT positive, 7 RDT and Plasmodium falciparum positive, and 1 suspected case, were collected and reanalysed. After application of the two molecular methods and sequencing, 291 cases including 245 P. falciparum, 15 Plasmodium vivax, 20 Plasmodium ovale, 6 Plasmodium malariae and 5 mixed infections (3 P. falciparum + P. ovale, 2 P. vivax + P. ovale) were confirmed. These patients had returned from Africa (95.53%) and Asia (4.47%). Although the prevalence displayed a small-scale fluctuation, the overall trend of the imported cases increased yearly.ConclusionsThese results emphasize the necessity of combined utilization of the four tools for malaria diagnosis in clinic and in field surveys of potential risk regions worldwide including Wuhan.

Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil

Pectobacterium brasiliense (Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different Pectobacterium and Dickeya species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other Pectobacterium and Dickeya species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 103 colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies.

One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/μL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients.

Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii (T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529bp (RE) sequence of T. gondii. The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×105 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment.

A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database.

The accurate and rapid identification of insect pests is an important step in the prevention and control of outbreaks in areas that are otherwise pest free. The potato-tomato psyllid Bactericera cockerelli (Šulc, 1909) is the main vector of 'Candidatus Liberibacter solanacearum' on potato and tomato crops in North America and New Zealand; and is considered a threat for introduction in Europe and other pest-free regions. This study describes the design and validation of the first species-specific TaqMan probe-based real-time PCR assay, targeting the ITS2 gene region of B. cockerelli. The assay detected B. cockerelli genomic DNA from adults, immatures, and eggs, with 100% accuracy. This assay also detected DNA from cloned plasmids containing the ITS2 region of B. cockerelli with 100% accuracy. The assay showed 0% false positives when tested on genomic and cloned DNA from 73 other psyllid species collected from across Europe, New Zealand, Mexico and the USA. This included 8 other species in the Bactericera genus and the main vectors of 'Candidatus Liberibacter solanacearum' worldwide. The limit of detection for this assay at optimum conditions was 0.000001ng DNA (~200 copies) of ITS2 DNA which equates to around a 1:10000 dilution of DNA from one single adult specimen. This assay is the first real-time PCR based method for accurate, robust, sensitive and specific identification of B. cockerelli from all life stages. It can be used as a surveillance and monitoring tool to further study this important crop pest and to aid the prevention of outbreaks, or to prevent their spread after establishment in new areas.

Identification of Small Berry Species in Food and Juice Using TaqMan-Based Real-Time PCR

Abstract Background: Public interest is growing for small berries in recent years because they are very delicious, low in energy, and full of bioactive compounds with potential health benefits. Similar to other food products, adulteration of small berry fruit products poses economic and safety problems to consumers. Objective: To protect consumers and regulate the small berry fruit products market, it is necessary to establish a robust method for detecting the authenticity. Methods: In this study, TaqMan-based real-time PCR assay was established for species identification of cranberry, raspberry, and blueberry to ensure authenticity of commercial small berry food products (pulp, dried fruit, fruit juice, jam, and puree). Results: Absolute detection limit was 0.1 pg/μL DNA for raspberries, 1 pg/μL DNA for blueberries, and 10 pg/μL DNA for cranberries. Practical LOD was 0.1% (v/v) for fresh juice. For processed juice, practical LOD was 1% for blueberry and red raspberries, 0.1% for black and yellow raspberries, and 5% for cranberry. Conclusions: The method was shown to be functional and effective to detect the raw material composition of cranberry, raspberry, and blueberry for commercial products. Highlights: TaqMan probe-based real-time PCR methods were designed to identify three small berries (blueberry, raspberry, and cranberry) in berry products. Efficient DNA recovery methods and detection strategy were established to ensure correct and sensitive testing of fresh small berries exhibited a detection limit of 0.1 to 10 pg/μL. The practical minimum detection levels were 0.1 to 5% in fresh and processed juice, including pasteurization and HTHP.

IL18 gene polymorphism and its influence on CD4+ T-cell recovery in HIV-positive patients receiving antiretroviral therapy

BackgroundPyroptosis has been reported to be critical in human immunodeficiency virus type 1 (HIV-1) pathogenesis and acquired immunodeficiency syndrome (AIDS) progression. Even after achieving viral suppression to undetectable levels during antiretroviral therapy (ART), exacerbated CD4+ T-cell death by pyroptosis has been suggested as one of the main causes of immunological non-response. Thus, variants in genes of pyroptosis pathway were studied in individuals with poor CD4+ T-cell reconstitution under antiretroviral therapy against HIV-1. Methods248 virologically suppressed ART-treated patients, 126 immunological non-responders (INR) and 122 immunological responders (IR) were recruited. Genotyping was performed using TaqMan probe-based realtime PCR platform. Genotype-guided flow cytometry analysis with general and recent thymic emigrant (RTE) CD4+ T-cells in pyroptosis was performed based on associated polymorphisms. ResultsBoth IL18 rs187238 G allele and GG genotype were associated as protection factors against poor CD4+ T-cell recovery (OR = 0.22; 95%CI = 0.50–0.77; P = .010 and OR = 0.58; 95%CI = 0.36–0.93; P = .022, respectively). It was demonstrated a statistical association between IL18 rs187238 genotypes of ART-treated patients and death by Caspase-1 levels (P = .020). The GG genotype showed lower pyroptotic RTE CD4+ T-lymphocytes levels in the ART-treated groups comparing with CC (P = .029) and CG (P = .018) genotypes, suggesting that the G allele presence may be related to a lower IL-18 production and thus reduced dead CD4+ T-cells levels by Caspase-1. ConclusionWe observed that IL18 G variant allele and genotype were associated with a better immunological response, which may influence on immunological recovery of HIV-positive patients receiving antiretroviral therapy, and low Caspase-1 activity levels was observed on GG genotype when compared CC genotypes.