Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers for phaC Gene of Bacillus megaterium

Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

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Rapid Identification of Measles Virus Vaccine Genotype by Real-Time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.01879-16 ◽

2016 ◽

Vol 55 (3) ◽

pp. 735-743 ◽

Cited By ~ 13

Author(s):

Felicia Roy ◽

Lillian Mendoza ◽

Joanne Hiebert ◽

Rebecca J. McNall ◽

Bettina Bankamp ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Measles Virus ◽

Rapid Identification ◽

Rt Pcr ◽

Outbreak Response ◽

Reverse Transcription Pcr ◽

Pcr Method ◽

Measles Outbreaks ◽

Genotype A

ABSTRACT During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.

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Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracisStrains

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3021-3028.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3021-3028 ◽

Cited By ~ 24

Author(s):

Michael J. Brumlik ◽

Urszula Szymajda ◽

Dorota Zakowska ◽

Xudong Liang ◽

Rajendra J. Redkar ◽

...

Keyword(s):

Bacillus Anthracis ◽

Long Range ◽

Repetitive Dna ◽

Related Species ◽

Molecular Level ◽

Repetitive Element ◽

Bacterial Genome ◽

Chromosomal Marker ◽

Closely Related Species ◽

Pcr Method

ABSTRACT The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105B. anthracis strains of diverse geographical origins. AllB. anthracis strains produced fingerprints comprising seven to eight bands, referred to as “skeleton” bands, while one to three “diagnostic” bands differentiated between B. anthracisstrains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracisstrains.

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Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes

Reproduction ◽

10.1530/rep.1.00088 ◽

2004 ◽

Vol 127 (2) ◽

pp. 201-205 ◽

Cited By ~ 30

Author(s):

Cai-Xia Yang ◽

Zhao-Hui Kou ◽

Kai Wang ◽

Yan Jiang ◽

Wen-Wei Mao ◽

...

Keyword(s):

Related Species ◽

Nuclear Transfer ◽

Blastocyst Stage ◽

Cell Nuclei ◽

Cell Stage ◽

Closely Related Species ◽

Copy Numbers ◽

Mitochondrial Dnas ◽

Morula Stage ◽

Pcr Method

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 × 104 copies of macaque mtDNA and 1.3 × 106 copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.

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THE SALIVARY GLAND CHROMOSOMES OF SIX CLOSELY RELATED BLACK FLIES NEAR EUSIMULIUM CONGAREENARUM (DIPTERA: SIMULIIDAE)

Canadian Journal of Zoology ◽

10.1139/z67-050 ◽

1967 ◽

Vol 45 (4) ◽

pp. 377-396 ◽

Cited By ~ 11

Author(s):

Robert W. Dunbar

Keyword(s):

Salivary Gland ◽

Related Species ◽

Chromosome Banding ◽

Black Flies ◽

Distinct Form ◽

Closely Related Species ◽

Banding Patterns ◽

Natural Group ◽

Y Chromosomes ◽

Giant Chromosome

The salivary gland chromosomes of nearctic black flies which form a natural group in Eusimulium close to E. congareenarum were analyzed in detail. Comparisons of their giant chromosome banding patterns disclosed six cytological segregates in two subgroups; subgroup A, with E. innocens, E. anatinum, E. congareenarum, and a cytologically distinct form near the latter designated E. congareenarum 'b'; subgroup B, with E. excisum and E. rivuli. Within each subgroup closely related species differ at least by (1) two or three interspecific inversions, (2) the intraspecific specific inversions present, and (3) the details of the X and Y chromosomes. The differences between the subgroups include (1) the position of the nucleolus, (2) the identity of the sex chromosomes as either the first or third pair, and (3) about 15 interspecific inversions between E. congareenarum and E. excisum, the most closely related species from either subgroup. The phylogenetic interrelationships have been traced by means of the interspecific inversions.

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