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ABSTRACT Schirmer strips and conjunctival swabs are used in ophthalmology for the collection of tears and fluids. One of the biggest challenges during the COVID-19 pandemic has been accurate diagnosis and, in some cases, ocular manifestations are among the first symptoms. In this context, this study aimed to collect evidence to support the use of Schirmer strips and conjunctival swabs as a method of sample collection for viral analysis. A literature search was conducted following the Scoping Review protocol defined by The Joanna Briggs Institute. Studies were analyzed regarding virus research, collection methods, and sample analysis. The findings support that viruses can be detected on the ocular surface through analysis of Schirmer strips and conjunctival swabs. However, additional studies with larger samples and time data are necessary to confirm these conclusions.
RESUMO A fita de Schirmer e o swab conjunctival são utilizados na oftalmologia como métodos de coleta para lágrimas e fluidos. Durante a pandemia da COVID-19, um dos desafios foi o diagnóstico correto e se sabe que, em alguns casos, as manifestações oculares podem ser um dos primeiros sintomas. Nesse contexto, este estudo tem como objetivo levantar evidência que destaque o uso de fitas de Schirmer e de swabs conjuntivais como método de coleta para análise viral. Conduziu-se uma revisão de literatura seguindo o protocolo para Scoping Review definido pelo Joanna Briggs Institute. Os pesquisadores analisaram os estudos em busca do vírus pesquisado, os métodos de coleta e os métodos de análise. Vírus podem ser detectados na superfície ocular através da análise de fitas de Schirmer e de swabs conjuntivais, entretanto novos estudos com populações maiores e com definições claras de tempo são necessários para conclusões mais assertivas no tema.
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Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.
Subject(s)
Humans , Polymerase Chain Reaction/methods , Nucleic Acid Synthesis Inhibitors , Molecular Diagnostic Techniques/methods , Spectrophotometry/instrumentation , Fluorometry/instrumentationABSTRACT
The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Subject(s)
Child , Humans , DNA Primers/chemistry , DNA, Bacterial/chemistry , Multiplex Polymerase Chain Reaction , Pneumococcal Infections/microbiology , Serotyping , Streptococcus pneumoniae/classificationABSTRACT
Objective To construct a recombinant eukaryotic expressing vector pcDNA-dec and provide a basis for further study on the bioactivity of decorin(DCN).Methods DCN cDNA was amplified by using polymerase chain reaction(PCR).Product of PCR and expressing vector were digested by restriction endonucleases,then ligated and transformed into JM109 bacteria.Results The specific DNA fragment was obtained by PCR as supposed.Product of PCR and expressing vector were digested by restriction endonucleases,then ligated to establish the recombinant eukaryotic expression vector pcDNA-dec.It was confirmed that DCN cDNA was inserted into the eukaryotic expression vector correctly by using digestion identification and sequencing.Conclusion The recombinant eukaryotic expression vector pcDNAdec of human DCN is successfully constructed.
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Objective To investigate effects of heroin on prolactin(PRL) mRNA in female rats.Methods Fifty female Wistar rats were randomly divided into five groups(10 rats each group): control,heroin-treated-3 d,heroin-treated-9 d,heroin-withdrawal-3 d and heroin-withdrawal-9 d groups.PRL mRNA of pituitary was assessed by(RT-PCR) and PRL concentration in rat plasma was determined by radioimmunoassay(RIA).Results The expression levels of PRL mRNA of pituitary were declined significantly in heroin-treated-3 d,9 d and heroin-withdrawal-3 d groups compared with control group(P0.05)).Conclusion The PRL mRNA in the pituitary and the PRL concentration in plasma in female rats are decreased by heroin and the effect can not be reversed in a short term after heroin withdrawal.
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Objective To develop a sensitive, specific, rapid and easy method for detecting E. coli O157 : H7 and other Shiga toxin-producing E. coli in food and clinical specimens. Methods A circular probe and capture probe specific for Shiga toxin-2 (stx2) gene had been synthesized and was used for determining the sensitivity of ramification amplification method (RAM). Different serotypes which contained stx2 gene, including an E. coli O157 : H7, an E. coli O46 : H38, an E. coli O111 : NM, an E. coli O22 : H8 and E. coli ATCC23716 (stx2 gene negative) were used for determining the specificity. All strains were detected by RAM to determine whether they contained stx2 gene. Real-time RAM was further studied to detect stx2 gene. Results The lowest number targets detected by RAM assay was 10 copies of stx2, indicating that RAM assay was as sensitive as conventional PCR. The result of specificity showed that different serotypes of strains were all positive for stx2 gene, while nonpathogenic E. coli ATCC23716 was negative. Among 32 isolates, 28 STEC isolates containing stx2 gene were positive by RAM assay, while others were negative. The RAM results were 100% in concordance with that of PCR. The real-time RAM results also showed that as many as 10 bacteria can be detected and the time of appearance of detectable signal was depended on the target concentration. Conclusion RAM assay can offer an alternative method for PCR to detect E. coli O157 : H7 and STEC in all types of specimens because of its simplicity and isothermal amplification.
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Objective To study the action of Mycoplasma genitulium (Mg) in pathogenesis of chronic non-bacterial prostatitis. Methods Mg culture and polymerase chain reaction (PCR) were perfomed on prostatic secretion specimens from 987 patients with chronic non-bacterial pristatitis and 125 healthy men. Results ①The positive rate of Mg in the prostatic secretion from the 987 patients was 31.5% (302/987). Among the 302 positive cases, 153 cases had other pathogens, of which Ureaplasma urealyticum (Uu) was the most (128). ②In the controls, the positive rate of Mg was 2. 53% (3/125). The difference between the two groups was statistically significant (X2=44. 32,P