and T7 RNA polymerase) were provided by KAINOS. Laboratories, Inc. (Tokyo, Japan). After the NASBA reaction, 90 mL of the hybridization solution was added.
Jpn. J. Infect. Dis., 68, 145–147, 2015
Short Communication
New Validated Rapid Screening Methods for Identifying Kudoa septempunctata in Olive Flounder (Paralichthys olivaceus) Yoshiko Sugita-Konishi1*, Yutaka Fukuda2, Koh-ichiro Mori3, Toru Mekata3, Toyohiko Namba4, Makoto Kuroda5, Akiko Yamazaki6, and Takahiro Ohnishi6 2Fisheries
1Graduate School of Environmental Health Science, Azabu University, Kanagawa 252-5201; Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602; 3National Research Institute of Aquaculture, Oita 879-2602; 4Incorporated Foundation Tokyo Kenbikyo-in, Tokyo 102-8288; 5Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo 162-8640; and 6Division of Microbiology, National Institute of Health Sciences, Tokyo 158-8501, Japan
SUMMARY: Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2–3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBANAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata. NAC), to screen infected fish during preshipment assessments and to screen imported fish. Real-time LAMP is a very commonly used rapid method in the microbiological field; it uses DNA information from the sample of interest (2). To develop a method that could be used to screen K. septempunctata, we established oligonucleotide sequences for a LAMP primer set (C3) for K. septempunctata detection, as shown in Table 1. C3 was constructed from a protein of K. septempunctata that was isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and treated with 0.1z triton X; this protein appeared to be a native protein of K. septempunctata spores. The C3 primer set did not cross-react with Kudoa thyrsites or Kudoa lateolabracis (data not shown). The real-time LAMP assay protocol used in this interlaboratory study was the following. DNA was extracted from 500 mg of frozen raw flounder muscle. Flounder muscle was suspended in 500 mL of 1.0 M NaOH and incubated at room temperature for 10 min in 1.5-mL tubes. Then, 500 mL of 1.0 M Tris-HCl (pH 8.0) was added, and the mixture was centrifuged at 20,000 × g for 5 min at 49 C. The supernatant (100 mL) was transferred to a fresh tube and mixed with 400 mL of distilled water. Real-time LAMP was then performed using a Genie II instrument (OptiGene, Horsham, UK) with the C3 LAMP primer set mixture and Isothermal Master Mix (OptiGene) containing a fluorescent intercalating dye. Real-time LAMP results were analyzed in terms of Tp values (time taken to generate a positive result). The reaction was run in a final volume of 25 mL; the reaction mixture contained 5.0 mL of DNA extract,
Since 2010, more than 250 cases of Kudoa septempunctata infection have been reported. The primary symptoms of foodborne diseases caused by K. septempunctata include diarrhea and emesis, which appear 2–20 h after ingestion. Symptoms are sometimes severe, although they usually disappear within 24 h. At present, 2 notification analytical methods are used in Japan. One method is a combination of quantitative real-time PCR and morphological detection using microscopy, which is adopted by the Ministry of Health, Labour and Welfare of Japan to identify causative agents and enforce public health procedures. The other method uses qualitative PCR, which is recommended by the Ministry of Agriculture, Forestry and Fisheries of Japan to prevent infected fish from entering the market (1). However, both these PCR methods are costly and time-consuming; thus, the development of a rapid and simple assay for screening is eagerly anticipated. Therefore, the aim of this study was to develop a new rapid, simple validated method. First, we developed 2 methods, real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBAReceived March 28, 2014. Accepted July 31, 2014. J-STAGE Advance Publication December 24, 2014. DOI: 10.7883/yoken.JJID.2014.133 *Corresponding author: Mailing address: Graduate School of Environmental Health Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan. Tel & Fax: ＋81-427-69-1887, E-mail: ykonishi＠azabu-u.ac.jp 145
Table 1. Sequence of the LAMP primers → 3′ ) Primer sequence (5′
Base
GCA GGA AGT TCA AAT TCA CGG TAA GGT TCA TGG AAG ACA CG TGT GCT CAA AGA CTC TCT TCG TCA CAT CGA CAT GTC TCT TCC TGG AGA CTA TTC TAC CGA TGA T TCT TAT CAG ACT TGT CCT GGA TCA CCG TAC TTA GTT TTG CGA ATG TGA CTT CAG ATG GAG TTC T
41 42 22 21 21 22
Primer name C3 LAMP primer set
C3_FIP C3_BIP C3_F3 C3_B3 C3_LoopF C3_LoopB
Table 2. Performance of each analytical method Qualitative PCR
NASBA-NAC
Real-time LAMP
Real-time PCR
Primer Cross reactivity
Ribosomal 28S DNA K. septempunctata only
Ribosomal 28S RNA K. septempunctata only
Kudoa spore protein (C3) K. septempunctata only
Time Instrument required
4h Amplification analyzer Electrophoresis
0.75 h Heat block
0.75 h Isothermal amplification analyzer
Ribosomal 18S DNA K. septempunctata K. thyrsites K. lateolabracis 2.5 h Real-time PCR analyzer
15 mL of Isothermal Master Mix, 5.0 mL of 5 × C3 LAMP primer set mixture (8.0 mmol/L each of FIP and BIP, 1.0 mmol/L each of F3 and B3, and 4.0 mmol/L each of Loop F and Loop B). This mixture was then incubated for 30 min at 659 C, along with real-time fluorescence monitoring. The other method, NASBA-NAC, combined a sensitive transcription-based isothermal nucleic acid amplification test (NASBA) and NAC, a simple and rapid visual detection method. The NASBA-NAC protocol for the inter-laboratory study was the following. A suspension of K. septempunctata was prepared by crushing 15 g of infected flounder muscle with 15 mL of phosphate-buffered saline. To extract nucleic acids including 28S rRNA from K. septempunctata, 100 mL of the suspension was vigorously mixed with 500 mL of the extraction reagent. Then, 2.5 mL of the extract was transferred to a new tube and mixed with 5 mL of the NASBA reagent that contained 28S rRNA specific primers for K. septempunctata, an internal control, and internal control specific primers. After incubation for 1 min at 459 C, 2.5 mL of NASBA enzyme was added to the tube, after which the NASBA reaction mixture was incubated for 30 min at 459 C. The extraction reagent, NASBA reagent (containing ribonucleotide triphosphates and dNTPs), and NASBA enzymes (containing Avian Myeloblastosis Virus Reverse Transcriptase, RNase H, and T7 RNA polymerase) were provided by KAINOS Laboratories, Inc. (Tokyo, Japan). After the NASBA reaction, 90 mL of the hybridization solution was added to the NASBA-reaction tube. Then, an NAC strip with both oligonucleotides immobilized on a membrane and latex-conjugated oligonucleotides was inserted into the tube. After 15 min, the NAC strip was evaluated visually. A sandwich-hybridized NASBA amplicon was detected as a blue line along the test line. This NASBANAC assay for K. septempunctata detection did not cross-react with K. thyrsites or K. lateolabracis. Table 2 shows the performance results of qualitative PCR (1), real-time PCR (3), and our NASBA-NAC and real-time LAMP assays. The NASBA-NAC and real-
time LAMP assays only required short procedure times, including pretreatments, compared with the procedure time required by the PCR methods. The PCR methods require a high cost with regard to instruments and operation, whereas the NASBA-NAC and real-time LAMP assays can generally be conducted using versatile instruments. Regarding cross-reactivity, real-time PCR was used to detect a broad spectrum of Kudoa spp. that parasitize flounders, as we could not exclude the possibility that Kudoa spp. other than K. septempunctata might be pathogenic. Except for real-time PCR, no methods validated in this study showed cross-reactivity with other Kudoa spp. As shown in Table 2, our results indicated that the new methods developed in this study were superior in terms of cost and time as compared with those of the notification analytical methods. Next, we conducted an inter-laboratory study to validate the new methods using 5 samples: 4 samples contaminated with K. septempunctata and 1 K. septempunctata-negative sample. These were used as duplicate blind samples. Evaluation was performed based on positive ratios in comparison with the notification methods, including qualitative PCR (5 laboratories) and real-time PCR (1 laboratory). Flounders infected with K. septempunctata (positive sample) and non-infected flounder (negative sample) were provided by the National Research Institute of Aquaculture, Oita Prefecture, Japan. The amounts of K. septempunctata in the positive samples were determined using real-time PCR and microscopic observations (4). The negative sample also was not infected with K. thyrsites or K. lateolabracis, as determined by PCR (1). For the test samples, pooled samples (15 g) were prepared by mixing 1 part of a flounder sample infected with 103–106 K. septempunctata spores/g with 29 parts of the negative sample. The results of the inter-laboratory study are shown in Table 3. The test samples, including a negative and 4 contaminated pooled samples with different amounts (3.3 × 102–1.1 × 105 spores/g) of contaminated flounder muscle, were distributed as duplicate blind 146
New Methods for Kudoa septempunctata Table 3. Validated test results Amount of K. septempunctata (spores/g)
Infected flounder Pooled sample1) Lab 1 Negative 104 4.0 × 104 5.8 × 105 3.4 × 106
Negative 3.3 × 102 1.3 × 103 1.8 × 104 1.1 × 105
0/2 2/2 2/2 2/2 2/2
2
3
4
5
Lab 1
0/2 0/2 0/2 0/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2
0/2 2/2 2/2 2/2 2/2
2
3
0/2 0/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2
Real-time PCR2) (copy no./g)
Real-time LAMP (positive sample no./ total sample no.)
NASBA-NAC (positive sample no./ total sample no.)
Qualitative PCR (positive sample no./ total sample no.)
4
5
Lab 1
0/2 2/2 2/2 2/2 2/2
0/2 2/2 2/2 2/2 2/2
0/2 0/2 0/2 2/2 2/2
2
3
4
5
0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 2/2 1/2 1/2 1/2 2/2 2/2 2/2 2/2
Lab 5 5.0 × 103 3) 5.9 × 104 5.6 × 105 1.8 × 106 3.0 × 107
1):
The infected flounder was mixed with the K. septempunctata-negative flounder in a ratio of 1:29. A copy number of more than 1.0 × 107 on real-time PCR or 1.0 × 105 spores/g is a violation of the Food Hygiene Law. 3): Since the sensitivity is superior to that of microscopic observation, real-time PCR is able to detect DNA of Kudoa spp. in the negative sample by microscopic observation. 2):
numbers (5). Because flounders infected with more than 106 spores (corresponding to 1.0 × 107 copy numbers/g using real-time PCR) are considered to violate the ``Food Sanitation Act'' of Japan, the sensitivity of realtime LAMP was suitable as a screening assay for enforcement. Confirmation using specimens infected with K. thyrsites and K. lateolabracis indicated that the specificity of these new methods was for K. septempunctata only. Thus, these methods can be used to make more rapid decisions than with the notification methods. In summary, in this study, we developed 2 new methods to simply and rapidly detect K. septempunctata in olive flounder. Based on the results of the interlaboratory study, the validity of these methods was confirmed. There were 3 benefits for using these methods. First, the methods could be conducted using pooled samples, which saves on labor. Second, a user can choose between real-time LAMP and NASBA-NAC, depending on the intended use. Third, genetic assays can be used to save time as compared with immunological assays, which are essential for establishing specific antibodies.
Fig. 1. (Color online) Pattern of NASBA-nucleic acid chromatography. During migration, when the sample is positive, the K. septempunctata-latex conjugated oligonucleotides hybridize with the K. septempunctata amplicons. The sandwich-hybridized NASBA amplicon was detected as a blue line along the test line on the nylon membrane. IC, internal control; r, reference; K.sp., K. septempunctata.
samples to 5 laboratories. These laboratories analyzed these samples using the new methods and qualitative PCR. One laboratory (Lab. no. 5) also used real-time PCR. The NASBA-NAC assay was able to detect 102 spores/g of K. septempunctata in the pooled samples (Fig. 1), which corresponded to the sensitivity with qualitative PCR. The positive predictive value (PPV) and negative predictive value (NPV) of NASBA-NAC were 100z and 0z, respectively, when the cut-off value was set at 102 spores/g in a pooled sample and the sensitivity was 3.3 × 102 spores/g. To examine commercial size flounders prior to shipping and juvenile flounders prior to farming, the NASBA-NAC assay appeared to be effective based on its sensitivity. In comparison, the real-time LAMP assay could not detect less than 104 spores/g of K. septempunctata in the pooled samples, although it clearly detected more than 105 spores/g in the pooled samples. When the cut-off value was set at 105 spores/g, the PPV and NPV results for real-time LAMP were 100z and 0z, respectively, and the sensitivity was 1.1 × 105 spores/g. In real-time PCR, the results are expressed as copy numbers/g of K. septempunctata. Because this method is able to detect both the sporogonic stage and presporogonic stage of K. septempunctata, it is difficult to convert these results to spore
Acknowledgments This study was partly supported by a Grant-inAid for food safety from Ministry of Health, Labour and Welfare of Japan.
Conflict of interest None to declare. REFERENCES 1. Grabner DS, Yokoyama H, Shirakashi S, et al. Diagnostic PCR assays to detect and differentiate Kudoa septempunctata, K. thyrsites and K. lateolabracis (Myxozoa, Multivalvulida) in muscle tissue of olive flounder (Paralichthys olivaceus). Aquaculture. 2012;338341:36-40. 2. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009;15:62-9. 3. Iijima Y, Nakanishi N, Furusawa H, et al. Inter-laboratory validation and applications of quantitative real-time PCR for the detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus). Jpn. J. Infect. Dis. 2012;65:436-8. 4. Kawai T, Sekizuka T, Yahata Y, et al. Identification of Kudoa septempunctata as the causative agent of novel food poisoning outbreaks in Japan by consumption of Paralichthys olivaceus in raw fish. Clin Infect Dis. 2012;54:1046-52. 5. Ohnishi T, Furusawa H, Yoshinari T, et al. Electron microscopic study of Kudoa septempunctata infecting Paralichthys olivaceus (olive flounder). Jpn. J. Infect. Dis. 2013;66:348-50.
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