Aplica����o da T��cnica de ���Nested Pcr��� Durante o Per��odo Pr��-Patente para Identifica����o de Schistosoma Mansoni no Hospedeiro Intermedi��rio Biomphalaria Glabrata

Biomphalaria glabrata, molusco de ��gua doce, desempenha um importante papel em Parasitologia M��dica, por ser o hospedeiro intermedi��rio de Schistosoma mansoni, trem��tode digen��tico respons��vel pela schistosomose intestinal. A detec����o de moluscos infectados pelo Schistosoma mansoni tem uma grande import��ncia em Sa��de p��blica, porque identifica focos de transmiss��o da schistosomose. As limita����es dos m��todos cl��ssicos para o diagn��stico de infec����es pr�� patentes fazem com que os m��todos de biologia moleculares sejam vistos como poss��veis alternativas atrav��s da detec����o de ADN do S. mansoni em moluscos hospedeiros. A detec����o de sequ��ncias espec��ficas de ADN por reac����o de polimerase em cadeia (PCR) tem-se verificado ser de extrema import��ncia para a an��lise gen��tica e diagn��stico de v��rias doen��as infecciosas. Neste estudo foi aplicada a t��cnica de Nested-PCR, com o objectivo de identificar, no per��odo pr��-patente, S. mansoni em moluscos expostos a 1, 5 e 10 mirac��dios em diferentes per��odos de tempo. Foram utilizados moluscos das estirpes albina e selvagem de B. glabrata. Para a realiza����o das t��cnicas de PCR e de Nested���PCR (NPCR) foram utilizados dois pares de oligonucle��tidos desenhados especificamente para detectar o ADN de S. mansoni . Verificou-se amplifica����o do fragmento de ADN do parasita em 80% das amostras analisadas, independentemente da dose de mirac��dios e do per��odo de exposi����o. O m��todo utilizado �� altamente sens��vel, mostrando ser uma ferramenta ��til na detec����o de hospedeiros intermedi��rios de S. mansoni, consequentemente na identifica����o de focos de schistosomose intestinal.

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Um protocolo de "nested-PCR" para detec����o do v��rus da anemia das galinhas

Este trabalho descreve o estabelecimento de um pro!tocolo de "nested-PCR" para a detec����o do v��rus da anemia das galinhas (CAV, chicken anemia virus), agente causador da anemia infecciosa das galinhas. Para a extra����o de DNA a partir de amostras cl��nicas um m��todo baseado no uso de tiocianato de guanidina mostrou-se mais sens��vel e pr��tico, do que os demais avaliados. Para a PCR inicial foi selecionado um par de primers que amplifica uma regi��o de 664 pares de bases (pb) do gene VP1. Para a "nested-PCR" propriamente dita, foi selecionado um segundo par que amplifica uma regi��o interna de 520 pb. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV. Outras trinta amostras v��rus e bact��rias, causadoras de doen��as em aves, n��o geraram produto de amplifica����o. A sensibilidade do teste foi determinada a partir de dilui����es seriadas de uma amostra vacinal de CAV. A "nested-PCR" mostrou ser mais sens��vel do que a PCR e foi capaz de detectar pelo menos 0,16 TCID50% da cepa vacinal. Al��m disso, detectou DNA viral em tecidos, soro e cama avi��ria de lotes com e sem sinais cl��nicos. Conclui-se que, como t��cnica para a detec����o do CAV, o protocolo de "nested-PCR" aqui descrito, �� mais sens��vel, r��pido e menos trabalhoso do que o isolamento viral em cultivo celular.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P&gt;0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeir��o Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A��, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Estabelecimento de um protocolo de Nested-PCR para detec����o do v��rus da anemia das galinhas e an��lise filogen��tica de cepas brasileiras.

O v��rus da anemia das galinhas (CAV) pode causar imunodepress��o em galinhas de todas as idades e doen��a nos frangos jovens, a qual �� caracterizada por severa anemia, atrofia da medula ��ssea e hemorragias. A doen��a cl��nica �� rara hoje, por��m a forma subcl��nica �� freq��entemente encontrada em cria����es comerciais e resulta num consider��vel decr��scimo do desempenho. O CAV apresenta variabilidade gen��tica, entretanto, em rela����o ��s amostras brasileiras do CAV, pouco ou quase nada desta variabilidade �� conhecida. O presente trabalho descreve um protocolo de Nested-PCR para a detec����o do CAV diretamente de amostras cl��nicas e analisa filogeneticamente as seq����ncias nucleot��dicas das amostras positivas buscando verificar se a patologia apresentada por estas est�� relacionada com a variabilidade gen��tica. Para a extra����o de DNA, o m��todo baseado em tiocianato de guanidina mostrou-se mais eficiente e pr��tico de executar que os demais testados. Foi selecionado um par de primers que amplifica uma regi��o de 664 pb do gene vp1 e outro par que amplifica uma regi��o interna de 539 pb para a realiza����o da Nested-PCR. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV e 30 diferentes isolados de v��rus e bact��rias causadoras de doen��as em galinhas, as quais n��o geraram produto de amplifica����o. A sensibilidade foi determinada a partir de dilui����es seriadas da vacina comercial para o CAV. A Nested-PCR mostrou ser mais sens��vel do que a PCR e foi capaz de detectar 0,16 DICC50% da cepa vacinal. Al��m disso, a Nested-PCR detectou DNA viral em tecidos, soro e cama avi��ria de lotes com e sem sintomas cl��nicos.O produto de amplifica����o de 539 pb do gene vp1 de 44 amostras, provenientes de diferentes Estados produtores de frangos do Brasil, foi seq��enciado e foram encontradas 10 novas seq����ncias nucleot��dicas do CAV. Estas 10 seq����ncias nucleot��dicas foram analisadas filogeneticamente pelo m��todo de dist��ncia neighbour joining com 1000 replica����es o qual, mostrou que n��o houve correla����o entre a patogenia apresentada nos animais e os grupos gen��ticos. Estas seq����ncias nucleot��dicas tamb��m foram comparadas com 30 cepas de CAV isoladas em outros pa��ses e n��o foi observada correla����o entre a distribui����o geogr��fica e a variabilidade gen��tica. Substitui����es de amino ��cidos foram observadas em 9 posi����es sendo que, 65R substituindo o res��duo Q e 98F substituindo o res��duo Y ainda n��o haviam sido observadas. Conclui-se que, como t��cnica de detec����o do CAV, o protocolo de Nested-PCR aqui descrito �� mais sens��vel e menos trabalhoso do que o isolamento viral. As amostras Brasileiras de CAV possuem caracter��sticas filogen��ticas similares ��s isoladas em outros pa��ses.

In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Detection of bovine herpesvirus 5 (BoHV-5) in formalin-fixed, paraffin-embedded bovine brain by nested PCR in Colombian cattle

Fifteen cases of viral meningoencephalitis in Colombian cattle were tested by nested PCR analysis for the detection of bovine herpesvirus 5 (BoHV-5). All fatal cases had shown severe neurological signs and had occurred following natural outbreaks of the disease. The neurological infection was histologically characterized by mild to moderate inflammatory changes in the brain and cerebellum, including meningitis, mononuclear perivascular cuffing, gliosis, haemorrhage, and the presence of Gitter cells (macrophages) accompanying large areas of malacia. No intranuclear inclusion bodies were seen in any of the cases. Results from BoHV-5 molecular extraction analyses showed there were five positive cases thus confirming the presence of the virus in Colombia.

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A��, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Detec����o do papilomav��rus humano (HPV) em carcinoma epiderm��ide de l��bio atrav��s da nested PCR e sua correla����o com os fatores de risco, caracter��sticas cl��nicas, anatomopatol��gicas e de sobrevida

P��s-gradua����o em Odontologia - FOA