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PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

ABSTRACT: The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) designing of primer pairs flanking specific internal sequences of 754 bp for P. intermedia sensu stricto and of ca. 1,100 bp for P. nigrescens, and (iv) synthesis (by PCR amplification) and digoxigenin labeling of quantities of DNA probes 754 and ca. 1,100 bp in size. The PCR-DNA probe assays combine either PCR amplification of a 754-bp specific sequence in the genomic DNA of strains of P. intermedia sensu stricto and hybridization with the 754-bp digoxigenin-labeled probe or amplification of a ca. 1,100-bp sequence of P. nigrescens and hybridization with the ca. 1,100-bp probe. Specific hybridization was observed with the amplified DNAs from 25 strains of P. intermedia and 24 strains of P. nigrescens, and no reaction was observed with the PCR products from 20 foreign species. The PCR-DNA probe assays described here should allow a highly specific and sensitive detection of P. intermedia sensu stricto and P. nigrescens in mixed infections.

SUBMITTER: Guillot E

PROVIDER: S-EPMC229862 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

Guillot E E Mouton C C

Journal of clinical microbiology 19970701 7

The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) d ...[more]

PMID: 9196214

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Project description:BackgroundEchinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency.MethodsIn the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method.ResultsThe multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/μl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/μl, 10 pg/μl and 5 pg/μl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms.ConclusionsThe developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.

Project description:Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

Dataset Information

PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

Publications

PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

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