SYBR Green-based Real-time PCR Research Articles

Human papilloma virus (HPV) and prostate cancer (PCa): The potential role of HPV gene expression and selected cellular MiRNAs in PCa development

BackgroundProstate cancer (PCa) is one of the most common and health-threatening cancers in men worldwide. The human papillomavirus (HPV) is considered one of the organisms with the potential to be involved in the progression of this cancer. In the present study, we evaluated the association between the expression levels of HPV genes with the expression of selected cellular miRNAs (miR-19a, miR-21, miR-23b, miR-34a, miR-150–5p, and miR-155) and their targets genes (P53, Rb, c-Myc, TIMP-1, MMP-2, MMP-9, PDCD4, Bcl-2, and Survivin) in PCa tissue samples. MethodsHPV detection and genotyping were performed on the tissues of 112 PCa patients and 39 healthy individuals. The expression profile of miRNA was evaluated by SYBR Green-based real-time PCR. As well Human Survivin ELISA Kit was utilized to determine the concentrations of Retinoblastoma, P53, survivin, Bcl-2, c-Myc, TIMP-1, MMP-2, MMP-9, and PDCD4 in the prostate tissues. ResultsAccording to our findings, HPV genome was detected in 28.7% (21/73) of PCa tissue specimens and 17.94% (7/39) control samples. There was no significant association between the presence of HPV infection with PCa (OR = 2.01, 95%CI = 0.8–5.68, P = 0.102). We found that mean expression level of miR-19a (3.7 ± 4.3, p-value: 0.0007), and −21 (2.5 ± 2.8, p-value<0.0001) were significantly higher and miR-23b (−2.14 ± 3.08, p-value: 0.003) and −34a (−3.12 ± 3.28, p-value: 0.0001) levels were significantly lower in PCa tissue samples than in control tissue samples. ConclusionPresent research indicated that HPV positive PCa has a distinct miRNA profile compared with HPV negative PCa.

NOVEL SPECIFIC PRIMERS FOR THE SPECIFIC DETECTION OF FUSARIUM OXYSPORUM F. SP. CUBENSE BASED ON SYBR GREEN REAL-TIME PCR

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt of banana worldwide. Foc is transmitted to another banana plantation via an infected banana sucker. A specific and sensitive detection assay could be used for disease-free propagule screening to prevent disease dispersal effectively. In this study, SYBR green-based real-time PCR assay was developed based on novel specific primers targeting the large subunit of RNA polymerase II (RPB1) gene. The partial RPB1 gene was amplified and sequenced from Foc isolate FOC1708 and SK3-2. Regions specific for Foc were identified and used for designing real-time PCR primers. The specificity of the designed primers was evaluated on genomic DNA from Foc isolates and non-target microorganisms. The results showed that the designed primers were specific to only Foc isolates from race 1 and tropical race 4 (TR4). The detection efficiency of the designed primers was compared to other published primers through optimized SYBR green-based real-time PCR assay and nested PCR. The new primers could detect the Foc genomic DNA at a minimum of 5 pg and target pathogen in a symptomless banana sucker. The specificity and sensitivity of the new primers were comparable to the published real-time PCR primers and the nested PCR assay. This developed assay with these novel primers can aid the disease quarantine for effective prevention and control of the Fusarium wilt of banana.

Standardization of SYBR Green-Based Real-Time PCR Through the Evaluation of Different Thresholds for Different Skin Layers: An Accuracy Study and Track of the Transmission Potential of Multibacillary and Paucibacillary Leprosy Patients.

The development of new molecular techniques is essential for the early diagnosis of leprosy. Studies in the field have failed to elucidate the performance of these tests in clinical practice. We aimed to design a new primer pair for the repetitive element (RLEP) target of Mycobacterium leprae and to test the accuracy of SYBR green-based real-time PCR through the evaluation of different thresholds for different skin layers. We also aimed to track the transmission potential of multibacillary and paucibacillary leprosy patients. The in vitro validation of our reaction resulted in a quantification limit of 0.03 bacilli. We then conducted a cross-sectional/cohort-based study of diagnostic accuracy. Patients were included, and skin samples were divided into four layers: epidermis, superior dermis, inferior dermis, and hypodermis. We also quantified M. leprae in nasal swabs of the included patients and compared the results to the number of household contacts also diagnosed with leprosy. One hundred patients with a clinical presentation compatible with leprosy were allocated to the leprosy or control group. Although the parasite load was greater in the superior and inferior dermis, M. leprae DNA was found in all skin layers. The best sensitivity was observed for the superior dermis using the presence of any quantifiable bacillus DNA as the threshold [sensitivity=59.26% (95% CI=45.97–71.32)]. In the epidermis, setting 1 quantifiable bacillus as the threshold resulted in 100% specificity (95% CI=92.29–100). The number of bacilli found in nasal swabs was not significantly related to the number of household contacts also diagnosed with leprosy. Paucibacillary patients tested positive only for bacillus fragments in nasal swabs but not for the entire bacilli. We can conclude that superficial biopsies might result in sensitivity loss, although different skin sample types will have little influence on the final accuracy. In contrast, threshold changes greatly influence these properties. Paucibacillary patients may not be a relevant source of disease transmission.

Establishment of TaqMan-based real-time PCR assay for rapid detection of duck circovirus.

Duck circovirus (DuCV) is widespread across the world and causes feather disorders in young ducks. It was identified as the causative pathogen of duck beak atrophy and dwarfism syndrome and primary sclerosing cholangitis. In this study, we aimed to establish a TaqMan-based real-time PCR assay to detect DuCV. The primers and probe were designed based on the conserved region of the DuCV Rep gene. After optimizing the reaction conditions, the minimum virus detection limit of the designed PCR technique was 39.4 copies/μL, 100 times that of conventional PCR (cPCR). No cross-reaction with six other common duck viruses was observed. The intra- and inter-assay variations were less than 1%. The detection rate of DuCV-positive clinical samples using TaqMan-based real-time PCR was higher than that using SYBR Green-based real-time PCR and cPCR. Collectively, these results showed that the established TaqMan-based real-time PCR detected DuCV with high sensitivity and specificity, and significant repeatability, making it suitable for clinical use. Hence, it may be used as a novel tool for the diagnosis and epidemiological investigation of DuCV.

NOVEL SPECIFIC PRIMERS FOR THE SPECIFIC DETECTION OF FUSARIUM OXYSPORUM F. SP. CUBENSE BASED ON SYBR GREEN REAL-TIME PCR

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt of banana worldwide. Foc is transmitted to another banana plantation via an infected banana sucker. A specific and sensitive detection assay could be used for disease-free propagule screening to prevent disease dispersal effectively. In this study, SYBR green-based real-time PCR assay was developed based on novel specific primers targeting the large subunit of RNA polymerase II (RPB1) gene. The partial RPB1 gene was amplified and sequenced from Foc isolate FOC1708 and SK3-2. Regions specific for Foc were identified and used for designing real-time PCR primers. The specificity of the designed primers was evaluated on genomic DNA from Foc isolates and non-target microorganisms. The results showed that the designed primers were specific to only Foc isolates from race 1 and tropical race 4 (TR4). The detection efficiency of the designed primers was compared to other published primers through optimized SYBR green-based real-time PCR assay and nested PCR. The new primers could detect the Foc genomic DNA at a minimum of 5 pg and target pathogen in a symptomless banana sucker. The specificity and sensitivity of the new primers were comparable to the published real-time PCR primers and the nested PCR assay. This developed assay with these novel primers can aid the disease quarantine for effective prevention and control of the Fusarium wilt of banana.

Molecular and microscopic prevalence of intestinal microsporidia among HIV+/AIDS patients in the Alborz province, Iran

Microsporidia are a large family of obligate intracellular protozoa; these medically important species are recognized as opportunistic agents in intestinal complications in HIV+/AIDS patients. The current cross-sectional study was designed and conducted from October 2018 to June 2019 to determine intestinal microsporidia in HIV+/AIDS patients by trichrome/Zeihl-Neelsen staining and SYBR Green-based real-time PCR. Out of 80 HIV+/AIDS patients, 23.75% (n=19) and 12.5% (n=10) were identified by molecular and microscopic methods, respectively. The predominant species in patients was Encephalitozoon (94%), which was found by quantitative real-time PCR and its high resolution melting tool. As far as we know, this is the first report from the Alborz region. The prevalence of intestinal microsporidiosis in this area in HIV+/AIDS patients was higher than both the global and national average. In addition to the need for further studies to prove protozoan pathogenicity in the aforementioned group, preventive measures should be considered.

Importance of Altered Gene Expression of Metalloproteinases 2, 9, and 16 in Acute Myeloid Leukemia: Preliminary Study.

Acute myeloid leukemia is a group of hematological neoplasms characterized by a heterogeneous course and high mortality. The important factor in the neoplastic process is metalloproteinases, proteolytic enzymes capable of degrading various components of the extracellular matrix, which take an active part in modifying the functioning of the cell, including transformation to cancer cell. They interact with numerous signaling pathways responsible for the process of cell growth, proliferation, or apoptosis. In the present study, changes in the expression of MMP2, MMP9, and MMP16 genes between patients with AML and people without cancer were examined. The impact of cytogenetic changes in neoplastic cells on the expression level of MMP2, MMP9, and MMP16 was also assessed, as well as the impact of the altered expression on the effectiveness of the first cycle of remission-inducing therapy. To evaluate the expression of all studied genes MMP2, MMP9, and MMP16, SYBR Green-based real-time PCR method was used; the reference gene was GAPDH. For two investigated genes MMP2 and MMP16, the lower expression level was observed in patients with AML when compared to healthy people. The MMP9 gene expression level did not differ between patients with AML and healthy individuals which may indicate a different regulation of gene expression in acute myeloid leukemia. However, no correlation was observed between the genes' expression of all tested metalloproteinases and the result of cytoreductive treatment or the presence of cytogenetic changes. The obtained results show that the expression of MMP2 and MMP16 genes is reduced while the expression of MMP9 is unchanged in patients with acute myeloid leukemia. This may indicate a different regulation of the expression of these genes, and possible disruptions in gene transcription or posttranscriptional mechanisms in the MMP2 and MMP16 genes, however, do not affect the level of MMP9 expression. Obtained results in AML patients are in contrary to various types of solid tumors where increased expression is usually observed.

Development of a rapid Salmonella detection method via phage-conjugated magnetic bead separation coupled with real-time PCR quantification

In this study, we used the lytic Siphovirus phage LPST10 to develop a rapid and accurate detection method for Salmonella. Salmonella cells were separated from complex food matrices through magnetic separation, then subjected to SYBR green-based real-time quantitative PCR detection. Bacterial concentrations of 102–106 (Colony forming unit per milliliter, CFU/mL), with 200 of μg LPST10 phage-MBs, and 15 min reactions at 37 °C were found to be optimal to allow the phage-magnetic beads to capture the Salmonella cells. The phage-magnetic beads specifically recognized Salmonella cells of different serotypes. Under optimal conditions, DNA was extracted from the resulting bacteria phage-magnetic bead complexes through either alkaline lysis with sodium dodecyl sulfate (SDS) or boiling. The detection assay was subsequently assessed in food matrices (Tryptic soy broth medium, milk, and lettuce). The detection limit reached <30 CFU/mL in the food matrices. The entire assay, including bacterial capture, DNA extraction, and real-time polymerase chain reaction (RT-PCR) detection, could be completed within 1.5 h. This approach using a lytic Siphovirus phage has potential for rapid, specific, and accurate detection of Salmonella enterica subsp.enterica in different food matrices.

Validation of Swab Sampling and SYBR Green-Based Real-Time PCR for the Diagnosis of Cutaneous Leishmaniasis in French Guiana.

Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of Leishmania species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold (CT ) was 24.4 (minimum CT , 17; maximum CT , 36) and was <35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.

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.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Abnormal expression of WWP1 in chronic lymphocytic leukemia and its clinical significance

目的检测E3泛素连接酶（WWP1）在慢性淋巴细胞白血病（CLL）患者中的表达，分析其与经典预后指标TP53、CD38、IGHV突变等的相关性及预后价值。方法48例初诊CLL患者肿瘤细胞WWP1的mRNA水平通过实时定量PCR（qPCR）检测，以9名年龄匹配的正常人作为对照组，分析其临床意义。结果对照组WWP1 mRNA表达中位数为0.007（95％CI 0.005～0.010），CLL组表达水平为0.031（95％CI 0.019～0.044），两者的差异有统计学意义（P<0.001）。WWP1相对高转录者和低转录者中位诊断至第一次治疗的时间分别为24个月和35个月，两者的差异有统计学意义（P＝0.022）。进一步亚组分析显示WWP1 mRNA表达水平与CD38表达和ZAP-70表达相关：CD38、ZAP-70阳性CLL患者较CD38、ZAP-70阴性患者的WWP1 mRNA表达水平显著升高（P值分别为0.012和0.029）。结论WWP1在CLL患者中存在异常高表达，而且与CLL临床预后因素ZAP-70和CD38的表达密切相关。

Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection

Porcine circovirus 4 (PCV4) is a novel circovirus first discovered in China in April 2019. Here, we established an SYBR Green I-based real-time PCR for quantitative detection of PCV4. A pair of specific primers was designed based on the conserved region of Cap of PCV4. The standard curve of the established real-time PCR.assay showed a good linear relationship. The sensitivity of the established real-time PCR was 100 times greater than that of conventional PCR, and the detection limit of the assay was 3 × 101 copies. There was no cross-reactivity with other swine DNA viruses, showing good specificity. The intra-group variation coefficient was 0.37–0.78 %, and the inter-group variation coefficient was 0.57–0.94%, indicating that the assay has good repeatability. Moreover, the analysis of clinical samples showed that the positive detection rate of PCV4 was 10.71% (18/168), while that of conventional PCR was 8.93% (15/168). Interestingly, co-infection with PCV2 or PCV3, or both PCV2 and PCV3, was also detected. In conclusion, the established SYBR Green I-based real-time PCR may be a cost-effective and rapid method for PCV4 clinical diagnosis.

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding species-specific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops.

DNA barcode based species-specific marker for Ocimum tenuiflorum and its applicability in quantification of adulteration in herbal formulations using qPCR

Ocimum tenuiflorum L. is a highly traded and extensively used species in Ayurvedic medicine owing to its immense therapeutic potential, which has led to adulteration of both formulations as well as traded raw drugs. To meet the standard international market requirements, quick and efficient techniques for authentication are required which can keep pace with the increasing demand for the herb. In view of this, a robust and accurate DNA marker for the qualitative and quantitative analyses of this species and derived products is reported. The species-specific markers (designated as CIM-PATH-fwd / CIM-PATH-rev) developed in this study was designed based on the differences in the nucleotide sequence of the psbA-trnH intergenic region. A consistent amplicon of 218 bp was generated across all the O. tenuiflorum accessions and was absent in other Ocimum species studied. Further, developed species-specific primers were assessed for simultaneous authentication as well as quantification of O. tenuiflorum in commercially purchased herbal formulations by SYBR green-based quantitative real-time PCR assay. The established qPCR method was determined to be effective for the authentication and relative quantitative assessments of the products. The species-specific marker developed can be successfully employed as a taxonomic marker for O. tenuiflorum.

Detection and molecular characterization of novel porcine bufaviruses in Guangxi province

Bufavirus (BuV) can infect a variety of hosts, including human, bats, rats, dog, swine and shrew species and are suggested related to diarrhea disease. Porcine bufaviruses (PoBuV) were first detected in Hungarian pig farms in 2016. To determine the prevalence and genetic diversity of PoBuV in China, we developed SYBR Green-based real-time PCR assays to detect PoBuV in Guangxi pigs. Real-time PCR detected PoBuV in 30 (29.13%, 30/103) of the samples with diarrhoeal intestinal tissues and rectal swabs. PoBuV-positive intestinal tissues and rectal swabs samples, co-infection with PEDV (15/30, 50.0%), followed by PDCoV (8/30, 26.67%), PoRV (6/30, 20.0%), PRRSV (5/30, 16.67%), and PCV2 (3/30, 10.0%) were observed. Fourteen complete genomes were cloned and sequenced. The results showed that they were 4189 bp in length and combined three open reading frames (ORFs) in the order 5′-NS1-VP1/VP2–3′. Fourteen strains shared 96.5%–99.8% identity among themselves and 92.7%–97.9% with the PoBuV reference sequences. Phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed fourteen strains belonging to PoBuV and were grouped into the three branches. These results help to provide new insight into the molecular epidemiology of PoBuV in the world.

SYBR Green-based real-time polymerase chain reaction assay for detection of porcine parvovirus 6 in pigs.

In this study, a SYBR Green-based real-time quantitative polymerase chain reaction (qPCR) assay was developed for rapid detection of porcine parvovirus (PPV) 6. Primer pairs targeting the conserved regions of PPV6 Capsid gene were designed. Sensitivity analyses revealed the lowest detection limit of the SYBR Green-based real-time PCR assay to be 47.8 copies/μL, which indicated it was 1000 times higher than that found in the conventional PCR investigations. This assay was specific and showed no cross-species amplification with other six porcine viruses. The assay demonstrated high repeatability and reproducibility; the intra- and inter-assay coefficients of variation were 0.79% and 0.42%, respectively. The positive detection rates of 180 clinical samples with SYBR Green-based real-time PCR and conventional PCR were 12.22% (22/180) and 4.44% (8/180), respectively. Our method is sensitive, specific, and reproducible. The use of SYBR Green-based real-time PCR may be suitable for the clinical detection and epidemiological investigation of PPV6.

Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA.

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

SYBR green-based real-time PCR detection of canine Babesia spp. in ixodid ticks infesting dogs in Kerala, South India

Ticks, being hematophagous ectoparasites, are notorious vectors that transmit an array of pathogenic viruses, bacteria, and protozoa while simultaneously exhibiting a zoonotic potential by transmitting pathogens that affect the health of owners in contact. The distribution of ixodid ticks of the genera Rhipicephalus and Haemaphysalis spp. in tropical climate of the state contributes to many serious tick-borne parasitic and rickettsial infections in domestic and wild canines. In south India, canine babesiosis is one of the most prevalent vector-borne parasitic diseases. Detection of parasites in tick vectors has significant epidemiological implications. The present study was designed to identify the presence of the most common vector-borne parasites of dogs in ixodid ticks using sensitive detection protocols. SYBR green-based real-time protocols detected Babesia vogeli in Rhipicephalus sanguineus sensu lato, while B. gibsoni was detected in R. sanguineus (s.l.) and Haemaphysalis bispinosa. The present study points out the need to reinvestigate the vectorial capacity of ticks in different geographical regions.

Symptom Severity of Nicotiana benthamiana Plants Inoculated with Agrobacterium Containing Infectious DNA-A Clones of Honeysuckle Yellow Vein Virus (HYVV)

To investigate the pathogenicity and virulence of the Honeysuckle yellow vein virus (HYVV) lacking betasatellites, PCR amplified unit-lengths of DNA-A genome of HYVV-[DJ] were cloned into binary vector pRI101-AN, and generated HYVV-[DJ]-1mer, -1.3mer and -2mer genomes. Each construct was transformed into Agrobacterium cells and agro-inoculated into young leaves of Nicotiana benthamiana. Except for the HYVV-[DJ]-1mer, HYVV-[DJ]-1.3mer and -2mer clones caused pronounced disease symptoms in N. benthamiana. HYVV-[DJ]-2mer agro-inoculated plants showed more severe plant stunting with downward leaf curling and crinkling than those of HYVV-[DJ]-1.3mer agro-inoculated plants. To discriminate the clone’s virulence quantitatively, SYBR Green-based real-time PCR was performed for the quantification of the target virulence gene DNA in agro-inoculated plants that were collected at weekly intervals for 4 weeks. Regression analysis was obtained from the standard curves by plotting Ct values over the logarithm of the amount of V1 protein gene DNA present in a dilution series of plasmid containing the full-length HYVV-[DJ] genome. Equation of the HYVV V1 DNA standard curve was used to quantify V1 gene DNA concentration in agro-inoculated plants with each clone. The accumulation of V1 gene DNA in HYVV-[DJ]-1.3mer agro-inoculated plants reached the peak level at 4 weeks post inoculation, while the accumulation of V1 gene DNA in HYVV-[DJ]-2mer agro-inoculated plants reached the peak level at 3 weeks post inoculation. The amount of V1 DNA in HYVV-[DJ]-1.3mer agro-inoculated plants was significantly more than that in HYVV-[DJ]-2mer agro-inoculated plants. Considering the results, there was a difference between the accumulation of virus DNA and the symptom severity of the analyzed plants agro-inoculated with each clone. It suggested that the infectious clones’ virulence is not necessarily correlated with the symptom severity.