Specific Detection of the Gene for the Extracellular Neutral Protease of Bacillus cereus by PCR and Blot Hybridization

1999 ◽  
Vol 65 (7) ◽  
pp. 3226-3228 ◽  
Author(s):  
H.-J. Bach ◽  
D. Errampalli ◽  
K. T. Leung ◽  
H. Lee ◽  
A. Hartmann ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Blot Hybridization ◽  
Neutral Protease ◽  
Gene Probe ◽  
Protease Gene ◽  
Specific Detection ◽  
Pcr Products ◽  
Group Members ◽  
Restriction Polymorphism

ABSTRACT A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of theB. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.

scholarly journals Identification and Classification of bcl Genes and Proteins of Bacillus cereus Group Organisms and Their Application in Bacillus anthracis Detection and Fingerprinting

2009 ◽  
Vol 75 (22) ◽  
pp. 7163-7172 ◽  
Author(s):  
Tomasz A. Leski ◽  
Clayton C. Caswell ◽  
Marcin Pawlowski ◽  
David J. Klinke ◽  
Janusz M. Bujnicki ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Length Polymorphism ◽  
Genomic Dna ◽  
Length Variation ◽  
Specific Detection ◽  
Bacillus Cereus Group ◽  
Closely Related Species ◽  
Length Polymorphisms ◽  
Pcr Products

ABSTRACT The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms.

Detection of Hepatitis a Virus and other Enteroviruses in Wastewater and Surface Water Samples by Gene Probe Assay

1991 ◽  
Vol 24 (2) ◽  
pp. 267-272 ◽  
Author(s):  
S. Dubrou ◽  
H. Kopecka ◽  
J. M. Lopez Pila ◽  
J. Maréchal ◽  
J. Prévot
Keyword(s):  
Surface Water ◽  
Cell Cultures ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Limit Of Detection ◽  
Blot Hybridization ◽  
Noncoding Region ◽  
Gene Probe ◽  
Dot Blot ◽  
Lower Efficiency

Enteroviruses were specifically detected by dot blot hybridization when using poliovirus type 1 (PV1) derived subgenomic radiolabeled cRNA probes (riboprobes) in environmental water specimens and in the cell cultures in which the viruses were amplificated. The riboprobe corresponding to the 5' noncoding sequence detected the majority of enteroviruses. Hepatitis A virus (HAV) was specifically detected by an HAV cRNA probe corresponding to the 5' noncoding region of its genome. By this test, the limit of detection of coxsackievirus B5 and echovirus 7 seeded in mineral water was 103 to 104 PFU/spot. In cell cultures, positive signals were observed in the lysates of cells infected by one PFU. Higher positive signals were obtained with a short PV1 probe (nt 221-670) corresponding to the 5' noncoding region, which is a well preserved sequence among the enteroviruses, than with PV1 genomic probe. Hybridization allowed a good detection of enteroviral RNAs in wastewater specimens, but with a lower efficiency in surface water. In this case, amplification of viruses in the cell cultures gave significant hybridization results.

scholarly journals In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

2014 ◽  
Vol 58 (3) ◽  
pp. 393-398 ◽  
Author(s):  
Tomasz Grenda ◽  
Elżbieta Kukier ◽  
Magdalena Goldsztejn ◽  
Krzysztof Kwiatek
Keyword(s):  
Clostridium Botulinum ◽  
Limit Of Detection ◽  
Corn Silage ◽  
Laboratory Examination ◽  
Specific Detection ◽  
Type D ◽  
Pcr Products ◽  
Type C ◽  
Examination Process ◽  
Feed Samples

Abstract A PCR-based procedure for detection of C. botulinum C and D in corn silage samples was validated. During the validation, method specificity, sensitivity, and accuracy were determined according to PN - EN ISO 16140:2004. Additionally, the specificity of the validated methods was proved by sequence analysis of PCR products obtained from examination of samples connected with botulism cases in cattle and mallard ducks. Limit of detection was estimated according to the Spearman - Kärber formula and expressed as LOD50. The obtained results showed that a 100% specificity was achieved. The sequencing of PCR products revealed 99% identity with sequences of bont/C and bont/D genes deposited in the GenBank. The sensitivity value ranged from 63.3% for C. botulinum type C to 75% for type D. The accuracy value varied from 72% for type C to 81.3% for type D. LOD50 was estimated at the levels of 0.272 (0. D 188-0395) spore/g for type C and 0.17 (0.1-0.289) spore/g for type D. The described PCR-based procedure enabled detection of C. botulinum C and D at the stage of liquid culture. This makes examination of feed samples possible without isolation process. The presented procedure could support the diagnosis of botulism by faster and specific laboratory examination process.

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