Development of a multiplex PCR assay for detection and identificationof a chickpea phytoplasma strain utilizing 16S rRNA and imp genesspecific primers

ABSTRACT A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis,Lactobacillus oris, and Lactobacillus reuteriand from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

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Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

Diseases of Aquatic Organisms ◽

10.3354/dao040177 ◽

2000 ◽

Vol 40 ◽

pp. 177-183 ◽

Cited By ~ 53

Author(s):

CR Osorio ◽

AE Toranzo ◽

JL Romalde ◽

JL Barja

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Photobacterium Damselae

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Identification ofClostridium beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricumisolated from silage, raw milk and hard cheese by a multiplex PCR assay

Journal of Dairy Research ◽

10.1017/s002202991200026x ◽

2012 ◽

Vol 79 (3) ◽

pp. 318-323 ◽

Cited By ~ 29

Author(s):

Paola Cremonesi ◽

Laura Vanoni ◽

Tiziana Silvetti ◽

Stefano Morandi ◽

Milena Brasca

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Raw Milk ◽

Most Probable Number ◽

Pcr Assay ◽

Promising Alternative ◽

Probable Number ◽

Multiplex Pcr Assay ◽

Microbiological Methods ◽

Hard Cheese

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation ofClostridiumspp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as:Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum.Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2–V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to bePaenibacillusandBacillusspp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

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Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v1i2.21506 ◽

2016 ◽

Vol 1 (2) ◽

pp. 38-42 ◽

Cited By ~ 4

Author(s):

Khairun Nessa ◽

Dilruba Ahmed ◽

Johirul Islam ◽

FM Lutful Kabir ◽

M Anowar Hossain

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Clinical Laboratory ◽

Proteinase K ◽

Microbiology Laboratory ◽

Pcr Assay ◽

E Coli ◽

Diarrheagenic Escherichia Coli ◽

Multiplex Pcr Assay ◽

Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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