RESUMO
ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
Assuntos
Humanos , Peste/microbiologia , DNA Bacteriano/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Magnetismo/métodos , Yersinia pestis/isolamento & purificação , Yersinia pestis/classificação , Yersinia pestis/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/química , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Separação Imunomagnética , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Magnetismo/instrumentaçãoRESUMO
Yersinia pestis é o agente causador da peste, doença primária de roedores, transmitida por pulgas infectadas, podendo infectar o homem e outros mamíferos. A maioria dos estudos de genotipagem realizada em cepas brasileiras de Y. pestis demonstrou baixo poder discriminatório, revelando padrões genotípicos semelhantes para cepas com diferentes características epidemiológicas. A análise do clustered regularly interspaced short palindromic repeats (CRISPR) vem sendo utilizada para genotipagem e estudos filogenéticos em diferentes gêneros bacterianos, inclusive de Y. pestis. Neste estudo foi realizada a genotipagem de cepas de Y. pestis da coleção de cultura do Serviço Nacional de Referência em Peste do Centro de Pesquisas Aggeu Magalhães - CPqAM/FIOCRUZ, isoladas nos três focos de peste do Estado de Pernambuco, pela análise dos locos CRISPR. Para as amplificações das 51 amostras, foram utilizados primers dirigidos às sequências CRISPR descritas na literatura (YPa, YPb e YPc). Os amplicons obtidos, após PCR, foram sequenciados, a fim de analisar a estrutura de cada loco CRISPR. Dois locos se apresentaram polimórficos: YPa, com alelos de 150pb a 570pb e YPb, com alelos de 330pb a 331pb. O loco YPc se mostrou monomórfico com alelos de 208pb. Os padrões CRISPR obtidos para cepas de Y. pestis de focos de outros países foram os mesmos observados nas cepas brasileiras. Diferentes arranjos dos espaçadores (YPa, YPb e YPc) foram observados e possibilitou que as cepas fossem agrupadas em 12 perfis genotípicos (PG1-PG12). Dois perfis foram distribuídos nos três focos estudados: PG1 perfil mais frequente (38 cepas) e PG3 (seis cepas). Os demais perfis foram específicos de uma determinada região de isolamento: PG2 para o foco de Triunfo e PG4-PG7 para o foco de Araripe. Os perfis PG8 a PG12 representaram o restante das cepas de focos de outros países. As mesmas cepas foram analisadas, anteriormente, através de onze locos VNTR (MLVA), e foram agrupadas em 35 perfis genotípicos
A menor diversidade observada no CRISPR ocorre, provavelmente, devido à região estar envolvida na codificação gênica e com maior pressão seletiva, portanto, com menor risco de sofrer mutação decorrente de estocagem. A análise dos locos CRISPR, complementar ao uso do MLVA, será útil na identificação e rastreamento de novas cepas, contribuindo para o estabelecimento de medidas de controle adequadas nas áreas de foco para prevenção da peste e na investigação de novos casos que possam surgir no Brasil
Assuntos
Humanos , Animais , Estudos Epidemiológicos , Sequências Repetitivas Dispersas , Peste , Yersinia pestis/classificação , Yersinia pestis/genética , Evolução Molecular , Variação Genética , Genótipo , Reação em Cadeia da Polimerase , Roedores , SifonápterosRESUMO
A plague episode in Surat in 1994, and its spread to other cities in India, lasted only a little over 2 weeks, but it created an unprecedented panic that had global repercussions. At first, the Surat hospital doctors could not diagnose the disease, but when they did, immediate intervention, in the form of preventation and treatment (administration of antibodies) prevented the disease from spreading beyond Surat, Delhi, Calcutta, Bombay and their vicinities. Fewer than 1,200 people were diagnosed with plague. A DNA-based study in 2000 decisively concluded that the Surat episode was a plague, but the Indian isolates were genetically more heterogeneous compared to others in the world.
Assuntos
Surtos de Doenças/história , História do Século XX , Humanos , Índia/epidemiologia , Peste/diagnóstico , Yersinia pestis/genéticaRESUMO
The pigmentation (pgm) locus is a large unstable area of the Yersinia pestis chromosome composed of a segment of iron acquisition (HPI) linked to a pigmentation segment. In this work we examined the mobility of HPI and the pigmentation segment in three Y. pestis isolates using successive subcultures on Congo red agar (CRA) plates. Strain P. CE 882 was shown to be highly stable while strains P. Exu 340 and P. Peru 375 dissociated into several phenotypes, PCR analysis showing evidence of changes in the pgm locus of the derived cultures. Strains P. Exu 340 and P. Peru 375 produced previously unreported cultures positive for the pesticin/yersiniabactin outer membrane receptor (psn+) but negative for the iron-regulated protein (irp2-), suggesting the occurrence of rearrangements in this chromosomal region and either a sequential loss or the loss of separated segments. These results provide evidence that besides deletion en bloc, specific rearrangements are also involved in the deletion events for that locus.
Assuntos
Pigmentação , Yersinia pestis/genética , Ilhas Genômicas , Ferro , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
The irp2 gene codes for a 190 kDa protein (HMWP2) synthesidez when highly pathogenic Yersinia are grown under conditions of iron starvation. In this work, the presence of irp2 in strains of Y. pestis isolated from different hosts during several plague outbreaks in the foci of Northeast Brazil wasstudied. For this purpose, 53 strains were spotted onto nylon filters and their DNA was hybridized with the A13 probe which is a 1 kb fragment of the irp2 coding sequence. All strains except two hybridized with the probe. However, when the initial stock culture of these two strains were analyzed, they both proved to bepositive with the A13 probe, indicating that the locus was lost after subculturein vitro but was always present in vivo. To examine the degree of conservation of the chromosomal fragment carrying irp2 among Brazilian strains, the hybridization profiles of 15 strains from different outbreaks, different hosts and different foci were compared. The hybridization profiles of these strains were all identical when their DNA was digested with either EcoRI, EcorRV or AvaII, indicatingthat the restriction sites surrounding the irp2 locus are very well conserved among Northeast Brazilian strains of Y. pestis. Altogether, these results suggest that the irp2 chromosomal region should be of prime importance for the bacteria during their multiplication in the host