RT-polymerase Chain Reaction Assay Research Articles

Chemokine (C-C motif) Ligand 6 Aggravates Hypoxia Reoxygenation-induced Apoptosis in H9c2 Cells Through Enhancing the Expression of Insulin-like Growth Factor 2-Antisense.

Chemokine (C-C motif) ligand 6 (CCL6), one of the small cytokines in the CC chemokine family, has been reported to involve in renal ischemia-reperfusion (I/R) injury. However, the role of CCL-6 in myocardial I/R injury is nonelucidated. In this study, we used in vitro H9c2 cell model to investigate the overall contributions of CCL6 to myocardial I/R injury. We found the elevated level of CCL6 from the reanalysis of data set GSE-4105 and in hypoxia-reoxygenation (H/R)-injured H9c2 cells. CCL6 silencing attenuated the cardiomyocyte apoptosis induced by H/R injury, whereas exogenous CCL6 treatment aggravated the apoptosis of H/R-injured H9c2 cells. During CCL6 administration, the expression of numerous long noncoding RNAs was differentially regulated. Quantitative RT-Polymerase chain reaction assay demonstrated that insulin-like growth factor 2 (IGF2)-Antisense (AS) had the highest induction by CCL6 addition. IGF2-AS silencing alleviated the apoptosis of H/R-injured H9c2 cells. Collectively, we have identified a potential mechanism by which high expression of CCL6 contributes to the H/R-induced apoptosis in H9c2 cells through enhancing the expression of IGF2-AS. These findings also give evidence of the feasibility of CCL6 or long noncoding RNA IGF2-AS as a potential target for modulation or therapeutic intervention in myocardial I/R injury.

Rapid Detection of Lily mottle virus and Arabis mosaic virus Infecting Lily (Lilium spp.) Using Reverse Transcription Loop-Mediated Isothermal Amplification

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65°C and 60°C for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

Behavioral and physiological processes in horses and their linkage with peripheral clock gene expression: A preliminary study

Abstract Behavioral and physiological processes have an innate 24-h cycle driven by the circadian master clock, which uses clock genes to generate rhythmicity and distribute temporal signals. Elucidating the blood gene expression in relation to the better known circadian rhythms in horses may contribute to improve the knowledge on the peripheral circadian rhythm control in this species. To do that, seven clinically healthy Italian saddle female horses were housed in individual boxes under natural photoperiod and environmental conditions. In each horse, locomotor activity was recorded continuously; blood samples and rectal temperature were recorded every 4 hours over a 48-hour period. To investigate the peripheral clock in horses, quantitative real-time RT polymerase chain reaction assays were designed to detected clock gene levels (Per 1, Per 2, and Cry 1) from blood samples. Blood cortisol serum level was also measured. Our results showed a daily expression of Per 1, Per 2, and Cry 1 in peripheral blood, associated with the daily rhythm of locomotor activity, rectal temperature and cortisol. In particular, a similar acrophase was observed for locomotor activity and Per 1; and for rectal temperature and Per 2. Rectal temperature and Per 2 also showed the same percentage of robustness of rhythm. We suggest the existence of a linkage between the peripheral clock genes Per 1 and Per 2 with locomotor activity and rectal temperature, although more studies are necessary to establish the exact mechanism of the peripheral clock.

Anti-dengue activity of Andrographis paniculata extracts and quantification of dengue viral inhibition by SYBR green reverse transcription polymerase chain reaction.

Background:Dengue virus is a leading cause of illness and death in the tropics and subtropics. As many as 400 million people are infected yearly. Dengue is caused by any one of four related viruses transmitted by mosquitoes. Currently, there is no vaccine to prevent infection with dengue virus and the most effective protective measures are those that avoid mosquito bites. When infected, early recognition and prompt supportive treatment can substantially lower the risk of medical complications and death. Nowadays, the search for natural plant products to fight against viral diseases has been increasing.Aims and Objective:To test the anti-dengu viral activity of both ethanolic & aqueous extract of Andrographis paniculata.Materials and Methods:In vitro antiviral activity were performed against dengue virus by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and SYBR green quantitative real-time polymerase chain reaction (PCR) method. Cytotoxicity was also evaluated by MTT. The dengue viral load (VL) inhibition in plant extracts was characterized by reverse transcription PCR (RT-PCR) analysis.Results and Discussion:In this study, the maximum nontoxic dose (MNTD) of A. paniculata plant was determined by testing the ethanolic extracts against Vero cells in vitro. Antiviral assay based on cytopathic effects denoted by degree of inhibition upon treating DENV 1–4-infected Vero cells with MNTD of A. paniculata has the most antiviral inhibitory effects. These results were further verified with an in vitro inhibition assay using MTT and RT-PCR, in which 55%–97% of cell viability were recorded in DENV-1–4-infected cells in different duration.Conclusion:Ethanolic extracts treated with dengue VLs also showed a significant changes which were reflected in RT PCR assay.

Dengue IgG Seropositivity and Zika Viral Load

BackgroundSecondary dengue virus (DENV) infections are typically more severe than primary infections. It is not known whether previous DENV infection is associated with higher Zika virus (ZIKV) quantitative RT-PCR results (viral loads (VLs)) in areas endemic for DENV such as Puerto Rico. Our objective was to analyze the association between previous DENV infection (DENV IgG-positive) and ZIKV VL among children with symptomatic ZIKV infection enrolled in the Sentinel Enhanced Dengue and Acute Febrile Illness Surveillance System (SEDSS) in Puerto Rico.MethodsThe study population for this analysis comprised individuals <18 years of age enrolled in SEDSS during 2016 who were ZIKV PCR-positive in serum (using the CDC Trioplex RT-polymerase chain reaction (RT-PCR) assay) within 5 days post-onset (DPO) of symptoms. ZIKV VLs (genome copies/mL) were determined using an RNA standard curve generated from the RT-PCR assay target amplicons. An in-house ELISA was used to ascertain the presence or absence of serum DENV IgG. Trends were assessed using Jonckheere-Terpstra and Chi-square for proportions tests. The Mann–Whitney-Wilcoxon test was used to compare medians. Linear regression modeling was used to determine the association between DENV IgG and ZIKV VL.ResultsOf the 319 individuals who met inclusion criteria, 163 have dengue IgG assays completed to date. Of these, 90/163 (55%) were DENV IgG-positive and 73/163 (45%) were DENV IgG-negative, and did not vary by sex (P = 1.00). However, the proportion of patients with DENV IgG-positivity increased with age (P < 0.001) (Figure). Overall, the median (interquartile range, IQR) ZIKV VL was 23,110 (7,452–84,003), and did not vary by age (P = 0.11) or sex (P = 0.33). However, the median ZIKV VL varied by DPO: 26,230 (DPO<3; n = 117), 15,159 (DPO≥ 3; n = 46), P = 0.002. The median (IQR) ZIKV VLs were: 24,073 (10,938–73,130) in DENV IgG-negative specimens and 22,658 (7,332–89,322) in DENV IgG-positive specimens (P = 0.91). Linear regression indicated no association between DENV IgG and ZIKV VL (P = 0.54).ConclusionDENV IgG-positivity increased with age among children with symptomatic ZIKV infection. ZIKV VLs did not vary by age, but decreased with increasing DPO. There was no association between DENV IgG and ZIKV VL.Disclosures All authors: No reported disclosures.

Toxoplasma gondii infection among chronic hepatitis C patients: A case-control study

To determine the detection rate of anti-Toxoplasma gondii (T. gondii) IgG and IgM in chronic HCV patients attending the Department of Tropical Medicine Mansoura University hospital in Egypt. This study included 120 adult chronic HCV patients, 81 decompensate cirrhosis (late-stage) and 39 chronic HCV non cirrhotic patients (early-stage) and 40 healthy blood donors as controls. Serum samples were examined for anti-Toxoplasma IgM and anti-Toxoplasma IgG antibodies by ELISA. Real-time RT-polymerase chain reaction assay was done for quantitation of hepatitis C virus. Anti-T. gondii IgG antibodies were detected in 75 (92.6%) of 81 late-stage cirrhotic patients, 30 (76.9%) of the 39 chronic HCV non cirrhotic patients (early-stage) and in 6 (15%) of 40 controls with statistically significant difference (P<0.001). Anti-T. gondii IgM antibodies were found in 11 (13.6%) in late stage patients, 5 (12.8%) in early stage and in 3 (7.5%) of controls with no statistical significant difference (P=0.610). There was no correlation between stage of fibrosis and IgM or IgG antibodies positivity in our studied groups (P=0.526). High IgG levels significantly correlated with high viral load (P=0.026). Our findings suggest that the serious opportunistic T. gondii infection represent a potential significant risk for chronic HCV patients. So, toxoplasmosis should be considered in their investigations and follow-up.

Pertussis Cases Hospitalised During The Winter-Fall-Summer Period Of 2010

Aim: The aim of this study is to present the epidemiological data of pertussis cases that were hospitalised, and monitored in Pediatric Infectious Diseases Ward of our hospital between February, and July 2010. Material and Methods: Nasopharyngeal smears were taken from all 26 patients who were admitted with pertussis- like syndrome between February and July 2010. These specimens were examined for B.pertussis by using RT PCR (real-time polymerase chain reaction) assay in the laboratory of Microbiology and Clinical Microbiology Department. Results: Pertussis PCR was found positive in 12 (46 %) patients. The median age of patients was 7 months, ranging between 40 days and 28 months. Fifty percent (n=6) of these patients was female and the other 50 % (n=6) was male. Most of the patients were admitted on May and June, 2010. The mean leucocyte and lymphosite counts were 17.200±11.700/mm3 (7.200-43.000/mm3 ) and 11.310±9.500/ mm3 (3.200-36.000/mm3 ), respectively. Two patients had CRP values &gt;5 mg/dL. All the remaining patients had normal levels of CRP. Father of 3 patients, mother of 4 patients, both parents of 2 patients and siblings of the 3 patients had a history of cough. An 18-month old patient required mechanical ventilation support for deep apnea. Another 3-month old patient who referred to the emergency department with hemiparesis started 2 weeks after the cessation of treatment, was diagnosed with acute disseminated encephalomyelitis (ADEM). Three patients were not vaccinated with DPT yet, because they were under 2 months old. A single dose of DPT vaccine was applied in 5 patients, 2 doses in 3 patients and 4 doses in 2 patients. Conclusions: Although pertussis vaccination exists in the routine vaccination protocol, whooping cough still remains as an important health problem. In order to protect infants from pertussis, parents, siblings and other individuals who had come in contact with the affected infants must have complete immunity against pertussis.

Isolation and Biological Characteristics of Chicken Adipose-Derived Progenitor Cells

Adipose-derived stem cells/adipose-derived progenitor cells (ADPCs) are multipotent stem cells that can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit, and other mammals but rarely from poultry. In this study, ADPCs were isolated from 1-day-old chicks. Primary ADPCs were subcultured to passage 15. The surface markers of ADPCs, CD29, CD44, CD71, and CD73, were detected by immunofluorescence and RT-polymerase chain reaction assays. The growth curves of different passages were all typically sigmoidal. In addition, ADPCs of different passages were successfully induced to differentiate into osteoblasts, adipocytes, and myocardial cells. The results suggest that the ADPCs isolated from chicken possess similar biological characteristics with those derived from other species, and their multilineage differentiation provides many potential applications.

Detection of Saint Louis Encephalitis Virus in Dengue-Suspected Cases During a Dengue 3 Outbreak

Arboviruses are frequently associated with outbreaks in humans and represent a serious public health problem. Among the Brazilian arboviruses, Mayaro virus, Dengue virus (DENV), Yellow Fever virus, Rocio virus, Saint Louis Encephalitis virus (SLEV), and Oropouche virus are responsible for most of human cases. All these arboviruses usually produce undistinguishable acute febrile illness, especially in the acute phase of infection. In this study we investigated the presence of arboviruses in sera of 519 patients presenting acute febrile illness, during a dengue outbreak in São José do Rio Preto City (São Paulo, Brazil). A multiplex-nested RT-polymerase chain reaction assay was applied to detect and identify the main Brazilian arboviruses (Flavivirus, Alphavirus, and Orthobunyavirus genera). The molecular analysis showed that 365 samples were positive to DENV-3, 5 to DENV-2, and 8 to SLEV. Among the positive samples, one coinfection was detected between DENV-2 and DENV-3. The phylogenetic analysis of the SLEV envelope gene indicated that the virus circulating in city is related to lineage V strains. These results indicated that during that large DENV-3 outbreak in 2006, different arboviruses cocirculated causing human disease. Thus, it is necessary to have an efficient surveillance system to control the dissemination of these arboviruses in the population.

Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi.

The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37% degrees C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RT D313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.

Real-time RT-PCR for quantitation of hepatitis C virus RNA

A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

Cloning and Expression Pattern of a Second [His5Trp7Tyr8]Gonadotropin-Releasing Hormone (Chicken GnRH-II) mRNA in Goldfish: Evidence for Two Distinct Genes

Complementary DNAs (cDNAs) encoding [Trp7Leu8]GnRH (sGnRH) and [His5Trp7Tyr8]GnRH (cGnRH-II) peptides have been isolated from the brain of goldfish (X. W. Lin and R. E. Peter, 1996, Gen. Comp. Endocrinol. 101, 282-296). In the present study we report the isolation of a second cDNA encoding cGnRH-II peptide in the brain of goldfish using reverse transcription (RT) and rapid amplification of cDNA ends. There is an overall 79.7% nucleotide sequence similarity between the two cGnRH-II cDNAs, with 65.3, 91.2, and 76.3% similarity between the 5'-untranslated regions, coding regions, and 3'-untranslated regions, respectively, of the two cGnRH-II cDNAs. Comparison of the two cGnRH-II precursors shows 87.2% amino acid similarity. The presence of two cGnRH-II genes was confirmed by the sequence analysis of the introns between exon II and exon III of the two cGnRH-II genes. Results indicate that the intron of the two cGnRH-II genes shows a high divergence in size and sequence, but contains the same splice junction. Expression of the two cGnRH-II mRNAs was detected by RT-polymerase chain reaction assay and Southern blot analysis in all five grossly dissected brain areas, olfactory bulbs and tracts, telencephalon, hypothalamus, optic tectum-thalamus, and posterior brain. However, there was a difference in apparent intensity of hybridization signal for the two cGnRH-II mRNAs in all brain areas, suggesting a difference of expression levels. sGnRH mRNA was detected in the olfactory bulbs, telencephalon, and hypothalamus, but not in midbrain and posterior brain areas. The present finding of duplicate cDNAs and genes for cGnRH-II in goldfish is in agreement with the recent tetraploidization in this species.