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Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species.

Arif M, Opit G, Mendoza-Yerbafría A, Dobhal S, Li Z, Kučerová Z, Ochoa-Corona FM - PLoS ONE (2015)

Bottom Line: The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity.All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species.The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Microbial Forensics & Food and Agricultural Biosecurity, 127 Noble Research Center, Oklahoma State University, Stillwater, Oklahoma, 74078, United States of America; Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, Oklahoma, 74078, United States of America.

ABSTRACT
Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics.

No MeSH data available.


Related in: MedlinePlus

Endpoint PCR sensitivity assays with individual primer set.Endpoint PCR performed with individual primer set using 10-fold serially diluted multi target artificial positive control (APC) starting at (A) 1 ng, (B) 100 pg, (C) 10 pg, (D) 1000 fg, (E) 100 fg, (F) 10 fg, and (G) 1 fg. Lane 1 to 5 are primer sets, ObsCo13F/13R (322 bp), PeaCo15F/14R (241 bp), BosCO7F/7R (184 bp), BruCo5F/5R (140 bp), and DecCo11F/11R (99 bp), respectively. Lanes M is a 1 kb ladder and W is a non-template control (water).
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pone.0129810.g003: Endpoint PCR sensitivity assays with individual primer set.Endpoint PCR performed with individual primer set using 10-fold serially diluted multi target artificial positive control (APC) starting at (A) 1 ng, (B) 100 pg, (C) 10 pg, (D) 1000 fg, (E) 100 fg, (F) 10 fg, and (G) 1 fg. Lane 1 to 5 are primer sets, ObsCo13F/13R (322 bp), PeaCo15F/14R (241 bp), BosCO7F/7R (184 bp), BruCo5F/5R (140 bp), and DecCo11F/11R (99 bp), respectively. Lanes M is a 1 kb ladder and W is a non-template control (water).

Mentions: Multiplex endpoint PCRs for detection of L. obscura, L. pearmani, L. bostrychophila, L. brunnea and L. decolor were performed using primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively (Fig 2 and S2 Fig). Each primer set specifically amplified the corresponding target Liposcelis species. The sensitivity of each primer set was checked with serially diluted APCs. Each primer set detected as little as 1 fg of target APC (Fig 3). The developed multiplex endpoint PCR gave positive results when tested against crude genomic DNA extracted from an individual insect (S2 Fig). No cross reactivity was observed against the non-targeted and/or closely related species of genus Liposcelis.


Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species.

Arif M, Opit G, Mendoza-Yerbafría A, Dobhal S, Li Z, Kučerová Z, Ochoa-Corona FM - PLoS ONE (2015)

Endpoint PCR sensitivity assays with individual primer set.Endpoint PCR performed with individual primer set using 10-fold serially diluted multi target artificial positive control (APC) starting at (A) 1 ng, (B) 100 pg, (C) 10 pg, (D) 1000 fg, (E) 100 fg, (F) 10 fg, and (G) 1 fg. Lane 1 to 5 are primer sets, ObsCo13F/13R (322 bp), PeaCo15F/14R (241 bp), BosCO7F/7R (184 bp), BruCo5F/5R (140 bp), and DecCo11F/11R (99 bp), respectively. Lanes M is a 1 kb ladder and W is a non-template control (water).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472718&req=5

pone.0129810.g003: Endpoint PCR sensitivity assays with individual primer set.Endpoint PCR performed with individual primer set using 10-fold serially diluted multi target artificial positive control (APC) starting at (A) 1 ng, (B) 100 pg, (C) 10 pg, (D) 1000 fg, (E) 100 fg, (F) 10 fg, and (G) 1 fg. Lane 1 to 5 are primer sets, ObsCo13F/13R (322 bp), PeaCo15F/14R (241 bp), BosCO7F/7R (184 bp), BruCo5F/5R (140 bp), and DecCo11F/11R (99 bp), respectively. Lanes M is a 1 kb ladder and W is a non-template control (water).
Mentions: Multiplex endpoint PCRs for detection of L. obscura, L. pearmani, L. bostrychophila, L. brunnea and L. decolor were performed using primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively (Fig 2 and S2 Fig). Each primer set specifically amplified the corresponding target Liposcelis species. The sensitivity of each primer set was checked with serially diluted APCs. Each primer set detected as little as 1 fg of target APC (Fig 3). The developed multiplex endpoint PCR gave positive results when tested against crude genomic DNA extracted from an individual insect (S2 Fig). No cross reactivity was observed against the non-targeted and/or closely related species of genus Liposcelis.

Bottom Line: The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity.All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species.The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Microbial Forensics & Food and Agricultural Biosecurity, 127 Noble Research Center, Oklahoma State University, Stillwater, Oklahoma, 74078, United States of America; Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, Oklahoma, 74078, United States of America.

ABSTRACT
Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics.

No MeSH data available.


Related in: MedlinePlus