Screening, identification and function of pPCP1 plasmid-derived sRNA in Yersinia pestis:a preliminary study / 军事医学

Objective To identify small non-coding RNAs encoded by plasmid pPCP1 and investigate their roles in biofilm formation, stress tolerance and/or virulence in Yersinia pestis.Methods Seven plasmid pPCP1-encoded sRNAs were identified by RNA-seq results in Y.pestis in our previous studies.Northern blot was used to validate the presence of the seven sRNAs.The sRNA-deletion mutants were constructed via λ-Red homologous recombination system.The biofilm formation, high salt tolerance and virulence of the phenotypes were compared between Y.pestis WT strain and sRNA mutants.Results and Conclusion The expression of seven pPCP1-encoded sRNAs was validated and the transcript length detected by Northern blotting corresponded to the length observed by RNA-seq.On this basis, five sRNA-deletion mutants were obtained.The capacity of biofilm formation was weakened upon deletion of sR3446.The tolerance of sR3446, sR3457, sR4338 and sR4340 mutants was found weakened in vitro compared to that of wild-type strain,but the tolerance of sR6143 was found increased.Slight virulence attenuation was found in two sRNA mutants ( sR4338 and sR4340 ) .The results suggest that pPCP1-deriving sRNA might be implicated in stress response, biofilm and virulence in Y.pestis.

Molecular approach for rapid diagnostic kit to detect Yersinia pestis from infected soil, water sample

Yersinia pestis is the cause for the acute infection and may be chosen for biological terrorism. Rapid diagnosis of this agent from infected soil - water is essential. Y. pestis habours 3 specific plasmids providing virulent factors to the bacterium. Objectives: (1) Testing the sensitivity and accurateness of PCR for Y. pestis. (2) Carrying out PCR using total genomic DNA serially diluted as a template. (3) Undertaking PCR on artificical experimentation by diluting Y. pestis in soil water as samples to test PCR based fast diagnostic approach. Methods: Yersinia pestis (inactivated) was used. Genomic DNA was extracted by DNeasy kit (Qiagen Inc). Using primer - pairs PLAF - PLAR (binding on pia gene of plasmid pPCP1) a specific product of PCR was 480 bp. After determination of the PCR sensitivity, a molecular based diagnostic kit was developed. Sensitivity and specificity of this kit was tested by PCR using diluted genomic DNA and bacterium itself; and mix of these templates in water and soil as samples. Results: With the diluted genomic DNA, it was successful to obtain specific PCR with 0.6ng template, which is equal to a single bacterium. Additionally, successful PCR amplification was obtained using the whole bacterium (without extraction of genomic DNA) and diluted quantity ranging from 101 to 102. Based on these results, the bacterium was artificially diluted with sample of soil - water as a natural isolate for PCR amplification. Conclusions: Evidently, approach for PCR-based diagnostic kit was successfully carried out from any template including soil - water samples with high fidelity, using the pia gene genetic marker of pPCR of Y. pestis.

Molecular identification of the Vietnamese yersinia pestis isolated in Vietnam using pla sequence of plasmid pPCP1 as genetic marker

Pla sequence composes of 480 nucleodides of pPCP1 plasmide originated from Yersinia pestis isolated from Viet Nam and collected by the technique of PCR of chemical line into TA vector and demonstrated in turn. PCR reaction gave specific result from 3 diversified sources for mould: total extracted ADN, ADN extracted from plasmide isolated from bacterium complete cell. A fast and accurate method of diagnosis was created basing on this operation. By BLAST programme for accessing Gene Bank, the pla-gene sequence of Yersinia pestis in Viet Nam is analogue to other strains worldwide