Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

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Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

Indian Journal of Animal Research ◽

10.18805/ijar.b-3774 ◽

2020 ◽

Author(s):

Jyoti Kumar ◽

G. G. Sonawane ◽

Fateh Singh ◽

S. Jegaveera Pandian ◽

Rajiv Kumar

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pasteurella Multocida ◽

Bacterial Species ◽

Sybr Green ◽

Economic Losses ◽

Conventional Pcr ◽

Specific Detection ◽

Pcr Assay ◽

Tissue Samples

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

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MOLECULAR INVESTIGATION OF HEMOTROPIC MYCOPLASMAS IN HUMAN BEINGS, DOGS AND HORSES IN A RURAL SETTLEMENT IN SOUTHERN BRAZIL

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652015000400014 ◽

2015 ◽

Vol 57 (4) ◽

pp. 353-357 ◽

Cited By ~ 6

Author(s):

Rafael Felipe da Costa VIEIRA ◽

Odilon VIDOTTO ◽

Thállitha Samih Wischral Jayme VIEIRA ◽

Ana Márcia Sá GUIMARAES ◽

Andrea Pires dos SANTOS ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Rrna Genes ◽

Human Beings ◽

Pcr Assay ◽

Factors Associated ◽

Human Sample ◽

Successful Amplification ◽

Species Specific

SUMMARY The aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanisalone, 12 ' Candidatus Mycoplasma haematoparvum' alone and 21 both). Only 1/100 (1.0%) human sample was positive by qPCR SYBR green, with no successful amplification of 16S rRNA or 23 rRNA genes despite multiple attempts. All horse samples were negative. Dogs >1 year of age were more likely to be positive for hemoplasmas ( p= 0.0014). In conclusion, although canine hemoplasma infection was highly prevalent, cross-species hemoplasma transmission was not observed, and therefore may not frequently occur despite overexposure of agents and vectors.

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