Journal of Stored Products Research 49 (2012) 184e188 Contents lists available at SciVerse ScienceDirect Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr PCR and isothermal-based molecular identification of the stored-product psocid pest Lepinotus reticulatus (Psocoptera: Trogiidae) Mohammad Arif a,b, Francisco M. Ochoa-Corona a,b,*, George P. Opit b,**, Zhi-Hong Li c, Zuzana Kucerová d, Václav Stejskal d, Qian-Qian Yang c a National Institute for Microbial Forensics & Food and Agricultural Biosecurity, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA b Department of Entomology and Plant Pathology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA c Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan, West Road, Beijing 100193, China d Crop Research Institute, Drnovská 507, 161 06 Prague 6, Czech Republic article info abstract Article history: Psocids of the genera Liposcelis and Lepinotus (Psocoptera) are well known small, soft-bodied stored- Accepted 3 February 2012 product pests that are difficult to identify using morphological characteristics, particularly the immature stages. Methods for quick, sensitive, and accurate identification of stored-product psocid species Keywords: belonging to these genera are required for identification, discrimination, pest management, and Lepinotus inspection, and quarantine of imported grain. A specific primer set, RetCO1F/RetCO1R, was designed by Molecular diagnostics targeting consensus sequences from multiple alignments of the CO1 gene of Lepinotus reticulatus for use SYBR Green qPCR in end point PCR, SYBR Green qPCR, and HDA. Lepinotus and Liposcelis species were tested to confirm the CO1 fi fi Isothermal amplification primer speci city. The described primer set allowed accurate identi cation of L. reticulatus, producing an amplicon of 119 bp in the three described assays. The primer set yielded a detection limit as low as 10 pg, 100 fg, and 1 ng using end point PCR, SYBR Green real time PCR, and HDA, respectively. All tests are accurate, rapid, sensitive, and useful for L. reticulatus identification and have multiple applications including biosecurity and forensic entomology. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction traditional morphological methods requires specialized knowledge and skills (Kucerová, 2002; Kucerová et al., 2009; Li et al., 2011). Psocids (Psocoptera) are commonly associated with human Accurate and timely identification and discrimination among dwellings, commodity stores, food and feed processing storage different psocid pests is important for effective integrated pest facilities, and museums containing animal specimens in the USA management (IPM). IPM programs for L. reticulatus would be and many other countries. Severe psocid infestations can cause facilitated by the availability of a reliable, accurate, and rapid serious weight loss and damage to stored grain (McFarlane, 1982; identification method. Although, to date, no studies have been Kucerová, 2002). Psocids also transmit pathogenic microorgan- published on the molecular identification of L. reticulatus,DNA- isms and contaminate food, leading to human health problems based diagnostic methods can supplement morphological anal- (Obr, 1978; Sidik et al., 1986; Kalinovic et al., 2006). A majority of ysis in the identification of this stored-product pest. the psocids infesting stored grains belong to two genera: Liposcelis Polymerase chain reaction (PCR) is an accurate and rapid (Liposcelididae) and Lepinotus (Trogiidae) (Opit et al., 2010). In method for insect detection and identification (Li, 2007; Li et al., North America, one of the psocid species that infests stored grain is 2011). Real-time PCR offers greater sensitivity and speed than end Lepinotus reticulatus Enderlein (Sinha, 1988; Throne et al., 2006). point PCR in the detection of targeted DNA (Zhang et al., 2007). The identification of psocid species at different life stages by TaqMan and SYBR Green qPCR, the two most popular of a variety of quantitative PCR (qPCR) formats and chemistries (Bustin, 2005), are * Corresponding author. Department of Entomology and Plant Pathology, 127 100 times more sensitive than end point PCR (Kim et al., 2008). The Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA. two assays offer comparable sensitivity and specificity (Maeda Tel.: þ1405 744 5527; fax: þ1405 744 6039. et al., 2003; Arikawa et al., 2008; Llorente et al., 2010), but the ** Corresponding author. E-mail addresses: [email protected] (F.M. Ochoa-Corona), low cost of SYBR Green qPCR makes this assay more suitable for [email protected] (G.P. Opit). routine monitoring than the TaqMan qPCR. 0022-474X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2012.02.001 M. Arif et al. / Journal of Stored Products Research 49 (2012) 184e188 185 Unlike end point PCR, SYBR Green qPCR eliminates the need for et al., 1994; Wilcox et al., 1997). The amplification of the CO1 post-PCR electrophoresis yielding a faster assay that minimizes regions was carried out in 20 ml volume reactions containing 0.4 mL unwanted residual waste. The implementation of such assays in Takara taq (5 U/mL) (Clontech Laboratories, Madison, WI), 1 mLof Extension and other applied programs is becoming increasingly each forward (Ron) and reverse (Nancy) primers (5 mM), 2 mL10X popular (Vincelli and Tisserat, 2008). More recently, helicase buffer, 1.2 mL MgCl2, 0.8 mL dNTPs (10 mM), 3 mL of DNA template, dependent amplification (HDA) and other isothermal amplification and 10.6 mL nuclease free water (Ambion, Austin, TX). PCR reactions strategies have been developed to amplify a targeted DNA segment were performed with a PTC-100 Peltier thermal cycler (MJ Research efficiently without the need for a thermocycler (Vincent et al., Inc., Waltham, MA). The cycling parameters were: Initial denatur- 2004; Tomlinson et al., 2010; Walker et al., 1992). Because HDA ation of 4 min at 94 C followed by 40 cycles at 94 C for 45 s, 41 C amplifies DNA at a single constant temperature by using a helicase for 60 s, 72 C for 90 s, and 7 min of final extension at 72 C. After enzyme to separate the double stranded DNA (Jeong et al., 2009), it amplification, 20 mL of PCR product were visualized in a 1.5 % is useful for amplifying target DNA under isothermal conditions in agarose gel in 1X TAE (Tris acetate EDTA) buffer. Amplified PCR a field or farm setting. In the present study, we report the devel- amplicons were purified using an illustra GFX PCR DNA and Gel opment of end-point PCR, HDA, and SYBR Green qPCR assays for Band Purification Kit (GE Healthcare Biosciences, Piscataway, NJ) identification of L. reticulatus using the multicopy cytochrome according to the manufacturer’s instructions and were directly oxidase subunit 1 gene (CO1). sequenced using an Applied Biosystems DNA Analyzer (Model # 3730) at the Oklahoma State University Nucleic Acids and Proteins 2. Materials and methods Core Facility. The Ron and Nancy primers were also used for sense and anti-sense strand sequencing. 2.1. Insects 2.4. DNA sequence analysis and primer design Liposcelis brunnea Motschulsky, Liposcelis paeta Pearman, Lip- oscelis fusciceps Badonnel, Liposcelis rufa Broadhead, Liposcelis CO1 gene sequences obtained with primers Ron and Nancy were obscura Broadhead, Liposcelis entomophila (Enderlein), Liposcelis aligned using CLUSTALX2 (Larkin et al., 2007) and were examined pearmani Lienhard, Liposcelis corrodens Heymons, Liposcelis bos- for regions of conservation. Percent identity matrices and nucleo- trychophila Badonnel, Liposcelis decolor (Pearman), and L. reticulatus tide sequence alignments were constructed using GeneDoc were reared at the Stored Product Entomology Laboratory, Okla- (Nicholas and Nicholas, 1997). Primers RetCO1F (50-TAG- homa State University, Stillwater, OK, on a mixture of 93% cracked TAAACTCCGGTGCAGGAA-30) and RetCO1R (50-TCTGATTCCTGC- hard red winter wheat, 5% Rice Krispies (Kellogg Company, Battle TAAATGAAGAGA-30) for L. reticulatus were designed from Creek, MI), and 2% wheat germ (wt/wt) in 360-ml glass canning jars a consensus sequence within the CO1 gene in L. reticulatus using fitted with mite-proof lids (Gautam et al., 2010). The top 3 cm of the Primer3 (Rozen and Skaletsky, 2000). Primer thermodynamics, Ò inner surface of each jar was coated with Fluon (polytetrafluoro- internal structures, and self- and heterodimer formation were ethylene; Northern Products, Woonsocket, RI) to prevent psocids examined in silico with mFold (Zuker, 2003). The specificity was accessing and gathering on the inside of the lid and/or escaping confirmed in silico by screening the primer sequences with BLASTn from the jars. Cultures were maintained at 30.0 Æ 1 C and 75 Æ 5% (Altschul et al., 1990). RH. Voucher specimens of 100 female L. rufa, L. pearmani, L. bostrychophila, L. decolor, L. entomophila, L. paeta, L. brunnea, 2.5. Nucleic acid amplification and cloning L. fusciceps, L. corrodens, L. obscura, and L. reticulatus, were depos- ited, in 95% ethyl alcohol, at the K. C. Emerson Entomology Museum 2.5.1. End point PCR at Oklahoma State University under lot numbers 101, 103, 106, 107, PCR assays were carried out in 20 mL reaction mixtures con- 110, 111, 114, 116, 118, 119, and 120, respectively. Lepinotus inquilinus taining 10 mL GoTaq Green Master Mix (Promega, Madison, WI), von Heyden and Lepinotus patruelis Pearman used were obtained 1 mL of each RetCO1F and RetCO1R primer (5 mM), 1 mLofDNA from Illinois State University, Normal, IL, USA. template, and 7 mL nuclease free water. PCR reactions were per- formed with an Eppendorf thermal cycler (Eppendorf, Hauppauge, 2.2. DNA isolation NY). The cycling parameters consisted of 35 cycles as follows: Initial denaturation for 3 min at 94 C followed by denaturation at 94 C DNA from 10e20 individuals for each species was isolated using for 20 s, annealing at 56 C for 30 s, extension 72 C for 30 s, and a Blood and Tissue Kit (QIAGEN, Valencia, CA) according to the final extension at 72 C for 3 min.