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.

Tobacco mosaic virus (TMV), Hosta virus X (HVX), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are a few of the major viruses that infect ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co-infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR (RT-PCR) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein (CP) genes of HVX and CMV, the nucleocapsid protein (NP) genes of TSWV and INSV, and the movement protein (MP) and CP genes of TMV. The primers were validated in vitro using single and multiplex RT-PCR assays. The detection limit of each primer set in multiplex RT-PCR was 100 fg (TMV), 1 fg (HVX), 10 fg (CMV), 10 pg (TSWV) and 10 pg (INSV). Forty-six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT-PCR. The multiplex RT-PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses.

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