PCR and Isothermal-Based Molecular Identification of The

Journal of Stored Products Research 49 (2012) 184e188

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Journal of Stored Products Research

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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.

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 1 C and 75 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 deposited, 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 performed 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. Positive and negative controls manufacturer’s instructions. The DNeasy Plant Mini Kit (QIAGEN) were included in each PCR amplification. A volume of 20 mLof was used to isolate DNA from dry cracked wheat grain according to amplified PCR product was electrophoresed in a 1.5% agarose gel in the manufacturer’s instructions. DNA from L. inquilinus and 1X TAE buffer. The amplicons were eluted from the agarose gel L. patruelis was isolated using prepGEMÔ (ZyGEM Corporation Ltd, using Quantum Prep Freeze N Squeeze Spin Columns (Bio-Rad, Hamilton, New Zealand). Plasmid DNA carrying the target sequence Hercules, CA) and cloned using the TOPO TA cloning kit (Invitrogen, for primer set RetCO1F/RetCO1R was purified from overnight Carlsbad, CA). The concentrations of total genomic and plasmid bacterial cultures using a QIAprep Spin Miniprep Kit (QIAGEN). DNA were determined using a NanoDrop v.2000 spectrophotom- eter (Thermo Fisher Scientific Inc., Worcester, MA). 2.3. Sequencing of CO I gene 2.5.2. Real time PCR The CO1 gene of L. reticulatus was amplified using degenerate Amplification with primer set RetCO1F/RetCO1R was carried out primers Ron (50-GGAKCACCTGATATAGCATTYCC-30) and Nancy (50- in 20 mL reaction mixtures containing 10 mL of Platinum SYBR Green CCCGGTAARATTAAAATATAAACTTC-30)(Simon et al., 1994). This set qPCR SuperMix-UDG (Invitrogen), 0.8 mL(5mM) of each RetCO1F of primers was used with modified PCR components and conditions and RetCO1R, 0.12 mL BSA (Invitrogen), 1 mL of template DNA, and to amplify approximately 440 bp within the CO1 gene, corre- 7.28 mL of nuclease free water. Positive and negative controls (non sponding to the mitochondrial DNA sequence region located template; water) were included in each round of PCR amplification between 1751 and 2191 bp in Drosophila yakuba (Burla) (Simon and each reaction was replicated three times. Cycling parameters 186 M. Arif et al. / Journal of Stored Products Research 49 (2012) 184e188 included two initial holds each for 2 min at 50 C and 95 C, 40 included in each round of PCR. The primers amplified the predicted cycles at 95 C for 12 s, and 60 C for 30 s. The assays were per- 119 bp amplicon of L. reticulatus within a range of melting formed in a Rotor-Gene 6000 thermocycler (Corbett Research, temperatures from 47.8 C to 62.1 C. Sydney, Australia). 3.3. Sensitivity assays 2.5.3. HDA Primers set RetCO1F/RetCO1R was used in 50 mL reactions using 3.3.1. End point PCR an IsoAmp II Universal tHDA Kit (New England Biolabs, Beverly, Use of primers RetCO1F and RetCO1R in end-point PCR yielded MA). Volumes of 25 mL of mix A and mix B were prepared sepa- a detection limit as low as 10 pg (10 2 ng), 100 pg (10 1 ng), and rately. Mix A contained 2.5 mL of 10X annealing buffer II, 0.75 mL 1000 pg (1 ng) with samples consisting of L. reticulatus DNA alone, (5 mM) of each RetCO1F and RetCO1R, 1 mL of template DNA, and L. brunnea extract spiked with L. reticulatus DNA, and cracked wheat 20 mL of nuclease free water. Reaction mix A was overlaid with extract spiked with L. reticulatus DNA, respectively. 50 mL mineral oil. Mix B contained 2.5 mL of 10X annealing buffer II, 2.5 mL of 100 mM MgSO2,4mL of 500 mM NaCl, 3.5 mL of each Iso- 3.3.2. SYBR Green real time PCR Amp dNTPs solution and IsoAmp Enzyme mix, and 9.5 mLof The SYBR green qPCR detected as little as 100 fg (10 4 ng) of nuclease free water. Each mix was gently mixed by pipetting fol- L. reticulatus DNA. The extracts of L. brunnea and cracked wheat lowed by brief centrifugation (4000 rpm for 5e10 s). Mix A was spiked separately with L. reticulatus DNA detected down to 100 fg incubated at 95 C for 2 min and immediately transferred onto ice. and 1 ng, respectively. Standard sensitivity assays using 10-fold Mix B was added to mix A and the two were mixed by pipetting and serially diluted L. reticulatus DNA and L. brunnea extract spiked incubated at 65 C for 90 min. The negative control consisted of the with DNA show R2 values of 0.99 (Fig. 1 and Table 1). Due to what same reaction mixtures containing nuclease free water instead of appears to be inhibitors, the R2 value of cracked wheat extract target template DNA. After amplification, 20 mL of amplified spiked with L. reticulatus DNA was 0.67 (Fig. 1C). Similarly, DNA product was electrophoresed in a 1.5% agarose gel in 1X TAE buffer. with concentrated cracked wheat extract was not detected. All The sizes of amplified DNA fragments were estimated with a 1 kb qPCR products amplified in the sensitivity assays (including the plus ladder (Invitrogen). spiking assays), were electrophoresed in agarose gels to check for differences in amplicon signal strength. A normalized fluorescence 2.6. Sensitivity and spiked assays value of 0.2 was chosen for comparing all standard curves gener- ated for the three sample types. The Ct values obtained in this way The sensitivity of each PCR assay was evaluated using 10-fold for L. reticulatus DNA alone were similar to those for L. brunnea dilutions of non-spiked and spiked DNA of L. reticulatus. Spiked extract spiked with L. reticulatus DNA, suggesting that L. brunnea samples contained rough insect macerate extract (supernatant) and extract has no inhibitory effect on PCR amplification by primers dry cracked wheat extract (supernatant). Dilutions of L. reticulatus RetCO1F and RetCO1R (Table 1). DNA ranged from 10 ng to 100 fg per reaction. For the first spiked sensitivity real time PCR assays, 1 mL of insect extract (one specimen of L. brunnea macerated in 50 ml of TE buffer) was added to each PCR tube containing 1 mL of diluted L. reticulatus DNA. In subsequent real time PCR assays, 1 mL healthy dry cracked wheat extract (one cracked wheat kernel macerated in 500 ml of TE buffer) was used in place of the insect extract. Each reaction was performed in three replicates.

3. Results

3.1. Primer design and thermodynamics

Because no L. reticulatus CO1 gene sequences have been deposited in the NCBI GenBank, neither the primers nor the amplicon met the desired 100% query coverage and 100% identity after an alignment with BLASTn. The predictions for internal structures, delta G (DG) plot values of 0.4 Kcal/mol of RetCO1F and 0.9 Kcal/mol of RetCO1R, were calculated using mFold.

3.2. Primer speciﬁcity

The primer set RetCO1F/RetCO1R was designed for use in end point PCR, real time PCR, and HDA. Primer specificity was tested against a near-neighbor exclusivity panel including L. inquilinus, L. patruelis, L. brunnea, L. bostrychophila, L. decolor, L. rufa, L. entomophila, L. paeta, L. fusciceps, L. obscura, L. pearmani, and L. corrodens. The primer set RetCO1F/RetCO1R showed no cross reactivity with any species in the exclusivity panel, and the expected 119 bp PCR product was amplified only from L. reticulatus. Fig. 1. Sensitivity of SYBR Green qPCR amplification of L. reticulatus DNA diluted in To further confirm the specificity, the primers were tested also a 10-fold series (starting at 10 ng/reaction), using the primer set RetCO1F/RetCO1R. (A) against DNA of the insect Homalodisca vitripennis (Germar) and of DNA alone, (B) insect extract spiked with DNA, and (C) cracked wheat extract spiked cracked wheat grain. Negative (water) and positive controls were with DNA. M. Arif et al. / Journal of Stored Products Research 49 (2012) 184e188 187

Table 1 Ct, M, and R2 values of real time PCR sensitivity assays obtained using DNA of Lepinotus reticulatus alone, extracts of Liposcelis brunnea, or dry cracked wheat spiked with L. reticulatus DNA, using the primer set RetCO1F and RetCO1R.

Template conc. SYBR real time PCR assays with per reaction DNA Insect extract Cracked wheat extract spiked with DNA spiked with DNA R2 e 0.99 0.99 0.67 Ex e 0.84 0.86 0.89 M e 3.77 3.72 3.62

Average Ct values 10 ng 20.47 21.31 21.78 of three replications 1 ng 24.42 25.58 30.35 100 pg 28.30 29.75 Errora 10 pg 32.46 33.41 Errora 1 pg 35.97 37.35 Errora 100 fg 39.09 39.71 Errora

a HV, very high error detected at the very end of the cycle; Ct, cycle threshold; R2, linear correlation; Ex, reaction efﬁciency; M, slope.

3.3.3. HDA occurred after concentrated cracked wheat extract was spiked with Ten-fold dilutions of L. reticulatus DNA were made down to 1 pg L. reticulatus DNA. Wheat seeds contain PCR inhibitors such as per reaction. Primer set RetCO1F and RetCO1R performed well DNAses, polyphenolic substances, and melanoidin, which can under isothermal amplification, yielding HDA sensitivity as low as interfere with the template amplification (Nakamura and Ohtsubo, 1 ng on agarose gel electrophoresis. 2010). Other assays performed with L. brunnea extract spiked with L. reticulatus DNA showed a 10-fold decrease in sensitivity in the end point PCR format but not in SYBR Green qPCR. 4. Discussion No PCR products were amplified from non-target insect species in the exclusivity panel, although all three assays successfully We report the development of assays using end-point PCR, SYBR amplified L. reticulatus DNA. L. reticulatus was the only member of Green qPCR, and HDA, all using a single pair of primers for the Lepinotus available to us for testing, but 10 species of the related fi identi cation of L. reticulatus. The multicopy CO1 gene of genus Liposcelis, which commonly infest the same commodities fi L. reticulatus was targeted to design primers for identi cation and together with L. reticulatus, also were included. high sensitivity. The COI gene has been shown to be reliable for the Most PCR assays for detection and identification of plant path- detection of L. entomophila (Yang et al., 2012). Optimal primers ogens require the use of a thermocycler (Kim et al., 2008). However, were designed in silico using Web interface applications. The soft- isothermal HDA offers significant advantages for diagnosis. In this ware program Primer3 was used to design primers, and mFold to investigation primer set RetCO1F and RetCO1R amplified target predict minimum free energies of self folding. Primers with delta G L. reticulatus DNA down to 1 ng under HDA isothermal conditions. D ( G) values equal or close to zero during PCR cycling enabled This method can be implemented easily at low cost in small labo- fi a robust reaction in which the PCR products were ampli ed in ratories or in grain and grain-product storage facilities, by operators a range of Tms from 47.8 C to 62.1 C. Thus, the primers are suit- lacking the specialized training required for PCR. able for use by different operators, in different laboratories, using Any of the three DNA-based detection and identification different PCR machines. methods described here can be used to supplement or confirm the PCR-based analyses are rapid and reliable (Muraji and Nakahara, morphological identification of L. reticulatus. The three methods fi 2002) for identi cation and discrimination of closely related can facilitate detection, confirm L. reticulatus identity, and be fi species and of insect species that are dif cult to identify morpho- applied during quarantine inspections and other pest control logically, as when they are immature (Qin et al., 2008; Sperling activities. The assays are practical, rapid and sensitive, low-cost, et al., 1994; Tuda et al., 1995; Litjens et al., 2001; Zapata et al., and efficient for the identification and discrimination of 2007). For example, PCR assays based on 16S rDNA genes were L. reticulatus. developed for the rapid discrimination and identification of Lip- oscelis species (Qin et al., 2008; Li et al., 2011). Other assays developed recently detect fungi (Llorente et al., 2010), bacteria (Pirc Acknowledgements et al., 2009), viruses (Chomic et al., 2011; Harper et al., 2011), and whiteflies (Zhang et al., 2007) using real time PCR. However, no real We thank Jacqueline Fletcher and Astri Wayadande, NIMFFAB, time PCR assay has yet been reported for rapid identification and OSU, for reviewing an earlier draft of this manuscript. We extend fi discrimination of psocids. our appreciation to E. Mockford, who con rmed the identity of the Nucleotide sequences based on the COI gene, diagnostic for psocid species used in the study and provided Lepinotus inquilinus L. reticulatus, are reported here for the first time. The sequences and Lepinotus patruelis. This work was funded by the Oklahoma Agricultural Experiment Station (project numbers OKL02695 and have been deposited online (GenBank accession # JN714839). Given the estimated number of undescribed insect species, espe- OKL02773), and by the Czech project MZE 0002700604. The mention of trade names or commercial products in this publication cially in the tropics, this will be useful in their identification. It will also be useful for determining whether populations of L. reticulatus does not imply recommendation or endorsement by Oklahoma State University. from different geographical regions are unique and can be unam- biquously discriminated and identified using molecular methods. Primers RetCO1F and RetCO1R detect as little as 10 pg of References L. reticulatus DNA after 35 cycles of real time PCR, but the assay is capable of detecting down to 100 fg after 40 cycles. Increasing the Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. Journal of Molecular Biology 215, 403e410. number of PCR cycles generates more PCR product (Cha and Thilly, Arikawa, E., Sun, Y., Wang, J., Zhou, Q., Ning, B., Dial, S.L., Guo, L., Yang, J., 2008. 1993) and enhances the overall sensitivity. Significant inhibition Cross-platform comparison of SYBR Green real-time PCR with TaqMan PCR, 188 M. 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