Rapid Screening Detection of Genetically Modified Papaya by Loop-Mediated Isothermal Amplification

Abstract

A loop-mediated isothermal amplification (LAMP)-mediated screening detection method for genetically modified (GM) papaya was developed targeting the 35S promoter (P35S) of the cauliflower mosaic virus. LAMP products were detected using a Genie II real-time fluorometer. The limit of detection (LOD) was evaluated and found to be ≤0.05% for papaya seeds. We also designed a primer set for the detection of the papaya endogenous reference sequence, chymopapain, and the species-specificity was confirmed. To improve cost-effectiveness, single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) system, which is a lateral flow DNA chromatography technology, was applied. LAMP amplification was clearly detected by the system at the LOD level, and a duplex detection of P35S and chymopapain was successfully applied. This simple and quick method for the screening of GM papaya will be useful for the prevention of environmental contamination of unauthorized GM crops.

INTRODUCTION

Papaya ringspot virus (PRSV) is a pathogenic plant virus in the genus Potyvirus that poses a threat to papaya plants found in tropical and subtropical regions. Papaya infected with PRSV exhibits symptoms such as leaf chlorosis, mosaic, and ringspots on the fruit, causing a marked reduction in the papaya harvests.¹⁾ To date, the most effective approach to prevent and control PRSV is to cultivate PRSV-resistant, transgenic papaya cultivars. Examples of such genetically modified (GM) papaya varieties have been developed in many countries and areas, including Hawaii (U.S.A.), China, Jamaica, Thailand, and Taiwan.²⁾ The first commercially available PRSV-resistant GM papaya, known as “line 55-1,” was developed in Hawaii and has now been authorized for food use in U.S.A., Canada, and Japan.^3,4) However, aside from this example, all other GM papayas have not yet been authorized not only for food but also for environmental release. Unauthorized GM crop contamination could have severely detrimental effects on both international trade and local economies.⁵⁾ Thus, there is a need for qualitative detection methods for unauthorized GM papaya. PCR tests has been already used as a general standard for GM organism (GMO) detection in worldwide.^6–9) With respect to unauthorized GM papaya lines, PCR-mediated specific detection methods have been developed,^10–14) however, PCR tests are time consuming and require expensive instruments and reagents. This means unauthorized GM papaya lines could be missed, as was the case for PRSV-YK, a line developed to resist the YK strain of PRSV that was found in some processed papaya products in Japanese markets; a cost-effective screening method to detect PRSV-YK could be extremely valuable.

Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific, and low-cost DNA amplification technology.¹⁵⁾ The amplification can be carried out at a constant temperature and does not require a thermal cycling step. Several LAMP-mediated GMO detection methods have been already developed and published.^16–19) It was reported that Loo et al. developed a LAMP-mediated GM papaya detection method using a microfluidic platform using microfluidic lab-on-a-disc.²⁰⁾

In this study, we developed a novel screening detection method for GM papaya with LAMP, targeting the 35S promoter (P35S) of the cauliflower mosaic virus and papaya endogenous gene, chymopapain.

The developed technique is highly specific and sensitive. For LAMP detection, we applied lateral flow DNA chromatography, and it makes possible for GM papaya detection not to use expensive instruments for routine GMO monitoring.

MATERIALS AND METHODS

Plant Materials

GM papaya line PRSV-YK mixing sample seeds were kindly provided by Fasmac Co., Ltd. (Kanagawa, Japan). For non-GM samples, maize and soybean seeds were purchased from Quality Technology International, Inc. (Elgin, IL, U.S.A.) and Ryokokushoji Co., Ltd. (Hiroshima, Japan), respectively. Seeds of the domestically harvested other crops such as rice and wheat were purchased at a market in Japan. Papaya seed of Hawaii Ouro was purchased over the internet and a papaya cultivar derived from Philippines was purchased at a grocery store in Japan.

DNA Extraction

Papaya genomic DNA was extracted with a GM quicker 2 (NIPPON GENE Co., Ltd., Tokyo, Japan) from papaya seeds according to the Ministry of Agriculture, Forestry and Fisheries (MAFF) standard method.²¹⁾ The concentrations of the extracted DNA solutions were measured by UV absorbance with an ND-1000 (NanoDrop Technologies, Wilmington, DE, U.S.A.), and the DNA solutions were diluted at 50 ng/µL. For each assay, 100 ng of genomic DNA was used.

Oligonucleotide Primers

The primers used in this study are listed in Table 1. A set of six primers containing two outer primers, two inner primers, and two loop primers was used for each LAMP amplification. The primer sets for target sequences were designed using LAMP Designer 1.13 (PREMIER Biosoft, Palo Alto, CA, U.S.A.). All primers were synthesized by Fasmac.

Table 1. The Oligonucleotide Primers Used for the LAMP Analyses

Target		Sequence
P35S	F3	5′-ATTGCGATAAAGGAAAGGC(C/T)ATCG-3′
	B3	5′-ACTTCCTTATATAGAGGAAGGGTC-3′
	FIP	5′-GAAGACGTGGTTGGAACGTCTTCTTAGTGGTCCCAAAGATGGA-3′
	BIP	5′-GCAAGTGGATTGATGTGATATCTCCTTGCGAAGGATAGTGGGA-3′
	LoopF	5′-TTTCCACGATGCTCCTCG-3′
	LoopB	5′-CGTAAGGGATGACGCACA-3′
Chymopapain	F3	5′-TCTACAATCTTGCTAACCCT-3′
	B3	5′-AGTCATCTTGAGAATAACCCAC-3′
	FIP	5′-TGGATGAAGGGAAATGGGAGGAGCAAGTAGAGTGAATGACAAGA-3′
	BIP	5′-CCACTCTTGAGAGTTAGCTAGTATGAGACATGTTGCCAAGAA-3′
	LoopF	5′-CGCATGGGTTTATATACTAGC-3′
	LoopB	5′-ACAATGGCTACGATGAGTTCA-3′

Preparation of a Positive Control Plasmid

Target sequence fragments from P35S and the endogenous chymopapain gene cloned and connected by the overlapping extension PCR, and then inserted into a pUC19 vector (NIPPON GENE). The plasmid was linearized and purified by a CsCl density gradient centrifugation. The concentration of the purified DNA was measured and diluted to theoretical copy numbers of 50000, 5000, 500, and 50 copies per reaction, with 5 ng/µL of ColE1 DNA (NIPPON GENE) solution. The plasmid was designated as pGMLP.

Preparation of Test Samples

For preparation of the mixed samples, the GM and non-GM papaya seeds were separately ground and mixed on a weight-to-weight basis. For the evaluation of the limit of detection (LOD), we used six mixing levels of test samples consisting of 0, 0.01, 0.05, 0.1, 0.5, and 100% PRSV-YK.

LAMP Assay

LAMP reactions were performed using a Genie II real-time fluorometer (OptiGene Ltd., Horsham, U.K.) as described previously.¹⁵⁾ The concentrations of primers for P35S detection were 0.2 µM for F3 and B3, 3.2 µM for FIP and BIP, and 0.8 µM for LoopF and LoopB. For the amplification targeting chymopapain, 1.6 µM of FIP and BIP was used instead of 3.2 µM. The LAMP rection were conducted using the Isothermal Master Mix (OptiGene), according to the manufacture’s protocol, and performed at 65 °C for 30 min, followed by annealing from 98 to 80 °C. LAMP amplifications were detected using fluorescent DNA binding dye. A template free control assay was also performed for all the primer sets. The assay was repeated 21 times for each target and each sample.

Signal Detection by Lateral Flow DNA Chromatography

The LAMP products were detected using lateral flow DNA chromatography. The reagents containing a developing solvent for single-stranded tag hybridization (STH) on a chromatography printed-array strip (C-PAS) and chromatography strips were purchased from TBA (Miyagi, Japan). We used the C-PAS4 membrane, on which four complementary tag sequences (A1, A2, A3, and A4), are linearly printed. In this study, A1 and A2 tag sequences were used. For P35S and chymopapain amplification, the 5′ end of the FIP and LoopF primers were tagged with an A1 and an A2 tag sequence, respectively. The LoopB primer was biotinylated for both targets. Signal detection by STH C-PAS was performed as described previously.¹⁷⁾ After LAMP amplification at the same conditions as used for the Genie II, each 1 µL of the LAMP reaction products was diluted by the developing solvent and the C-PAS4 membrane was dipped into the mixture. Blue line(s) would appear after 10–15 min visually.

Duplex Detection by Lateral Flow DNA Chromatography

The STH C-PAS system was used for the duplex detection of two target sequences, P35S and chymopapain. As mentioned above, the FIP primer was tagged with an A1 tag sequence for P35S detection, and the LoopF primer was tagged with an A2 tag sequence for chymopapain detection. Both LoopB primers were biotinylated. For LAMP amplifications, equal concentrations of both primer sets were mixed. The analyses were repeated 21 times for LOD evaluations.

RESULTS AND DISCUSSION

The Specific Detection of P35S and Chymopapain to PRSV-YK by LAMP Assay

The Genie II system was used for the fluorescence detection of the targets as it is a lightweight instrument, making it suitable for real-time isothermal amplification detection of LAMP products both in the laboratory and in the field. In our previous studies, we designed a specific primer set for P35S detection with GM maize and soybean.^16,17) It has been reported that P35S was introduced into several GM papaya lines containing PRSV-YK.^10,22) Hence, the specificity of the primer set to PRSV-YK was checked; correct amplifications were observed from PRSV-YK, with no unexpected amplifications detected in the template free control (data not shown) and the non-GM papaya. We also designed a new primer set targeting the papaya endogenous sequence chymopapain gene,²³⁾ which has been used previously for a PCR-mediated detection method. For GMO detection, endogenous species-specific sequences are required as internal positive controls. The specificity was evaluated using other plant genomic DNAs such as maize, soybean, rice, and wheat. The amplifications of chymopapain were only detected for papaya genomic DNAs (Fig. 1A), confirming that the primer set was specific. We also used other two papaya varieties such as Hawaii Ouro and a papaya cultivar derived from Philippines to evaluate the specificity of the primer set targeting chymopapain, and amplifications appeared from three papaya samples (Supplementary Fig. 1).

Fig. 1. Representative Results of for LAMP Analyses with Genie II

(A) The specificity tests for chymopapain amplification. Genomic DNAs derived from non-GM (0%) papaya, 100% papaya, non-GM maize, non-GM soybean, rice, wheat seeds were used as templates. (B) LOD evaluation targeting P35S. 0% or 0.05% GM papaya were used as templates. The amplification profiles are shown in (A), (B), and annealing curves are shown in (C), (D).

LOD Evaluation of the Detection Method by LAMP Assay

To evaluate the LOD of the developed method using the Genie II system, mixed samples prepared from GM and non-GM papaya seeds were used. We have applied the criterion for LOD evaluation for qualitative analyses as being ≥20 times positive in 21 repeats, meaning that the false-negative rate should be within 5%.^16,24) The LOD evaluations are summarized in Table 2, which shows that the LOD for GM papaya was ≤0.05% and >0.01%. It was reported that the LOD of the PCR-mediated PRSV-YK detection method was 0.1%,²⁵⁾ meaning that the developed method was equal or more sensitive than the one. The representative results are shown in Fig. 1B. The LAMP amplification appears in less than 15 min even for the 0.05% GM papaya samples. The annealing curve analyses were conducted for the LAMP products. The annealing temperature is specific for each target sequence, and such an analysis is useful for confirmation of the specificity of the LAMP products. Single peaks were detected in both chymopapain and P35S (Figs. 1C, D). These results suggested that the primer sets were indeed specific.

Table 2. LOD Evaluation Targeting P35S for GM Papaya

GMO amount	100%	0.5%	0.1%	0.05%	0.01%	0%
Positive/total	21/21	21/21	21/21	21/21	14/21	0/21
Detection rate	100%	100%	100%	100%	66.7%	0%

Development of a Positive Control Plasmid for GM Papaya Detection

For GM detection, a reliable reference material is indispensable as a positive control to ensure the quality of the test results. However, it is quite difficult to obtain unauthorized GM crops as a control sample. Instead, plasmid DNA that includes the target sequences have sometimes been used in Japan. Thus, we developed a positive control plasmid, pGMLP, for GM papaya detection. The pGMLP had 355 bp of the chymopapain segment and 248 bp of the P35S segment, tandemly connected and cloned into the vector (Fig. 2). The positive control plasmid was purified and diluted as a copy number ratio. In total, the papaya genome size was considered to be 372 Mbp per haploid genome.²⁶⁾ The theoretical copy number of GM papaya in 100 ng DNA at 0.05% can be estimated as 124 copies per reaction. When we evaluated the serial dilution sample of the positive control plasmid, it was revealed that 50 copies were enough for detection (Table 3).

Fig. 2. Schematic Diagram (A) and Sequences (B) of Integrated Fragments Inserted into Plasmid pGMLP

Primers of chymopapain and P35S for LAMP amplifications are indicated by gray and black arrows, respectively.

Table 3. Detection Rates for Diluted pGMLP

Copy number	50000	5000	500	50
P35S	21/21	21/21	21/21	21/21
Chymopapain	21/21	21/21	21/21	21/21

Specific Detection of P35S and Chymopapain to GM Papaya by LAMP Assay with STH C-PAS

In the pursuit of more cost-effective detection, we applied a STH C-PAS system. The STH C-PAS is a lateral flow DNA chromatography technology. For P35S and chymopapain detection, the FIP and LoopF primer were tagged with A1 and A2 tag sequences, respectively, and both the LoopB primers were labeled with a biotin. In the C-PAS membrane, capture sequences which are complementary tag sequence are printed. After LAMP amplification, the C-PAS was dipped into the mixture containing the LAMP reaction. The LAMP products are then trapped with hybridization between tag and capture sequences on the membrane. In the STH C-PAS system, three red lines are printed and used as positional markers. The bottom line was used for differentiation between signals derived from A1 and A2 tags. Signal(s) appear as blue line(s) and be detected visually. In our previous study, the LODs of LAMP products with STH C-PAS were almost equal to those obtained using the Genie II system.^16,17) As shown in Fig. 3A, 0.05% of GM papaya are detectable with C-PAS. This suggests that the LODs for GM papaya detection with LAMP using C-PAS are at least as good as those obtained using the Genie II system. The blue lines above the top red line such as lane 2 in Fig. 3A are flow control lines. Amplifications targeting chymopapain were observed from all samples including 0% GM papaya (Fig. 3B). To improve the efficiency, rapidity and simplicity of the analytical step, we attempted a duplex LAMP detection using the system. We mixed the primer sets of P35S and chymopapain and found that both targets were clearly detected even at the LOD level (Fig. 3C). The signal derived from chymopapain could therefore be used as a control line for a practical inspection.

Fig. 3. Representative Results for LAMP Analyses with STH C-PAS

(A), (B) Lanes 1 is 50 copies of pGMLP, and lanes 2, 3, and 4 are 0, 0.05, and 100% GM papaya, respectively. (C) Duplex detection for LAMP products with the STH C-PAS system. Lanes 1, 2, and 3 are no template, 0 and 0.05% GM papaya, respectively. The A1 and A2 tag sequences were used for P35S and chymopapain detections, respectively. The blue lines in A1 and A2, which are binding sites of A1 and A2 tags, indicate P35S and chymopapain detections, respectively.

CONCLUSION

In conclusion, we developed a novel screening method for GM papaya that can be used for the detection of unauthorized GM lines, including PRSV-YK in Japan. The method uses LAMP-mediated screening, targeted to P35S and the endogenous reference sequence, chymopapain, to achieve species-specific results with a LOD of ≤0.05%. More cost-effective detection was developed using STH C-PAS, which is a lateral flow DNA chromatography technology. Current PCR-mediated PRSV-detection method is robust and reliable, but it takes more than 2 h for run time. On the other hand, the developed method requires a half hour for LAMP amplification, and the amplification can be detected using C-PAS without any expensive instruments such as real-time PCR. This technology is then cheaper than PCR and other available screening tools and allows for more rapid screening.

Acknowledgments

We would like to thank Fasmac Co., Ltd. for supplying weight-based mixed samples for GM papaya. This study was conducted under the research project on “Regulatory research projects for food safety, animal health and plant protection,” JPJ008617, 21454496 funded by the Ministry of Agriculture, Forestry and Fisheries of Japan.

Conflict of Interest

Y. Kagiya was an employee of Fasmac Co., Ltd. and S. Futo is President of Fasmac Co., Ltd. Y. Minegishi is an employee of NIPPON GENE Co., Ltd. The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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