Species-specific Polymerase Chain Reaction Primers Research Articles

Acquisition and transmission of Grapevine fanleaf virus (GFLV) by Xiphinema index and Xiphinema italiae (Longidoridae).

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Rapid detection of CITES-listed shark fin species by loop-mediated isothermal amplification assay with potential for field use

Shark fin is a delicacy in many Asian countries. Overexploitation of sharks for shark fin trading has led to a drastic reduction in shark population. To monitor international trade of shark fin products and protect the endangered species from further population decline, we present rapid, user-friendly and sensitive diagnostic loop-mediated isothermal amplification (LAMP) and effective polymerase chain reaction (PCR) assays for all twelve CITES-listed shark species. Species-specific LAMP and PCR primers were designed based on cytochrome oxidase I (COI) and NADH2 regions. Our LAMP and PCR assays have been tested on 291 samples from 93 shark and related species. Target shark species could be differentiated from non-target species within three hours from DNA extraction to LAMP assay. The LAMP assay reported here is a simple and robust solution for on-site detection of CITES-listed shark species with shark fin products.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

ABSTRACTPufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish

Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish

Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max). Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction) primer set and seed soaking (without DNA extraction) for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence) and one naturally infected seed in a 300-seed sample (0.33 % incidence). The PCR-based assay was rapid (< 9 h), did not require DNA extraction and was very sensitive.

Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control

A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 105 CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.

In vitro biofilm forming capacity on abiotic contact surfaces by outbreak-associated Vibrio harveyi strains

Objective: To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi (V. harveyi) strains. Methods: Thirty six Gram-negative V. harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay. Phenotypic characterization of mucoid biofilm producing V. harveyi strains were screened on Congo red agar, thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar, respectively. Results: Only five V. harveyi strains (14%) were mucoid biofilm producers characterized by formation of black colonies, whereas the remaining 31 strains (86%) were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre. The morphological, physiological and biochemical characteristics of these isolates were studied using standard protocols. Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V. harveyi 16S rRNA sequence. All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested. V . harveyi strains isolated from various seafood were able to form biofilms of different capacity, and the strains VB267, VB238 and VB166 isolated from cat fish, shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates. Conclusions: It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene. These properties allow these bacteria to survive, proliferate and persist in street vended seafood outlets.

English

Megasphaera micronuciformis is an anaerobic microbe isolated from humans. However, since the microbe is strictly anaerobic, its cultivation requires complicated facilities, making detection costly. For rapid, inexpensive detection and identification of M. micronuciformis in the clinical setting, a new technique is necessary. This study aimed to develop a species-specific PCR primer set for the detection of M. micronuciformis. A ribosomal DNA-specific PCR primer Mm2F was designed for M. micronuciformis. Analytical specificity data showed that the PCR primer set Mm2F/Mega-X produced amplicons from M. micronuciformis but not from the other species tested, including 4 Megasphaera species and representative related species. Of the 52 oral samples from Japanese subjects evaluated in our study, 71% were positive for M. micronuciformis, suggesting the likelihood that M. micronuciformis is widely distributed in the oral cavity of the Japanese population.&nbsp; &nbsp; Key words: Megasphaera micronuciformis, specific polymerase chain reaction (PCR), Mm2F, oral bacteria.

Development and application of tuf gene-based PCR and PCR-DGGE methods for the detection of 16 Bifidobacterium species

A total of 16 Bifidobacterium species were assayed by polymerase chain reaction (PCR) and PCR–denaturing gradient gel electrophoresis (PCR–DGGE) methods targeted on a 770-bp region of the tuf gene. Based on this sequence, a genus-specific primer set and 12 primer sets for 12 Bifidobacterium species including those previously reported for six probiotic species were developed. On the other hand, when these 16 Bifidobacterium species were subjected to PCR–DGGE analysis, 13 product migration patterns were obtained. PCR products for strains in pairs of B. adolescentis/B. thermophilum, B. longum/B. magnum and B. lactis/B. gallinarum migrated the same distance on the DGGE gel. Combined with species-specific PCR primers specific to B. adolescentis, B. longum and B. lactis, all of the 16 Bifidobacterium species could be identified. In addition, the subspecies of B. animalis, i.e., B. animalis and B. lactis, could be discriminated. This study indicated that the tuf gene is highly useful for the molecular detection of different Bifidobacterium species. Using the PCR and PCR–DGGE methods, 16 Bifidobacterium species, including those from probiotic products and those from other origins, could be rapidly identified.

Identification of Veillonella tobetsuensis in tongue biofilm by using a species-specific primer pair

Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae have been reported to be isolated from human oral cavities. The recently detected Veillonellatobetsuensis in human tongue biofilms was proposed as a novel Veillonella sp.In this study, to determine the distribution and frequency of V. tobetsuensis, we established a method for the detection and identification of V. tobetsuensis by using polymerase chain reaction (PCR) using a species-specific primer pair. The primer pair for V. tobetsuensis was designed on the basis of the nucleotide sequence of the 70-kDa heat shock protein (dnaK) gene of V. tobetsuensis JCM 17976T (=ATCC BAA-2400T). The primer pair generated a specific PCR product for V. tobetsuensis but not for other oral Veillonella spp. With the PCR procedure using the primer pair, we could detect less than 10 ng of genomic DNA extracted from V. tobetsuensis. Thus, the PCR method using this primer pair is suitable for the specific detection and identification of V. tobetsuensis. The distribution and frequency of V. tobetsuensis were investigated by PCR using this species-specific primer pair. V. tobetsuensis was detected in 5 of 27 subjects. V. tobetsuensis was recovered from 19% (5/27) of subjects with other Veillonella species. And, prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in these subjects. V. tobetsuensis is likely to coexist with other Veillonella spp. in tongue biofilm.In this study, the species-specific PCR primer pair for V. tobetsuensis was designed using partial sequences of the dnaK gene. This is the first report using a species-specific primer pair for PCR to determine the distribution and frequency of V. tobetsuensis in tongue biofilm.

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species-specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. The microbiological composition of foods was analysed by molecular methods. Using species-specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species.

Evaluation of conventional PCR for detection of Strongylus vulgaris on horse farms

Strongyle parasites are ubiquitous in grazing horses. Of these, the bloodworm Strongylus vulgaris is regarded as most pathogenic. Increasing levels of anthelmintic resistance in strongyle parasites has led to recommendations of decreased treatment intensities, and there is now a pronounced need for reliable tools for detection of parasite burdens in general and S. vulgaris in particular. The only method currently available for diagnosing S. vulgaris in practice is the larval culture, which is laborious and time-consuming, so veterinary practitioners most often pool samples from several horses together in one culture to save time. Recently, molecular tools have been developed to detect S. vulgaris in faecal samples. The aim of this study was to compare the performance of a conventional polymerase chain reaction (PCR) assay with the traditional larval culture and furthermore test the performance of pooled versus individual PCR for farm screening purposes. Faecal samples were obtained from 331 horses on 18 different farms. Farm size ranged from 6 to 56 horses, and horses aged between 2 months and 31 years. Larval cultures and PCR were performed individually on all horses. In addition, PCR was performed on 66 faecal pools consisting of 3–5 horses each. Species-specific PCR primers previously developed were used for the PCR. PCR and larval culture detected S. vulgaris in 12.1 and 4.5% of individual horses, respectively. On the farm level, eight farms tested positive with the larval culture, while 13 and 11 farms were positive with the individual and pooled PCRs, respectively. The individual PCR method was statistically superior to the larval culture, while no statistical difference could be detected between pooled and individual PCR for farm screening. In conclusion, pooled PCR appears to be a useful tool for farm screening for S. vulgaris.

Development of Three Additional Culex Species-Specific Polymerase Chain Reaction Primers and Their Application in West Nile Virus Surveillance in Canada

In 2002, more than 17,000 mosquito pools collected in Canada (Quebec, Ontario, and Manitoba) were tested at the National Microbiology Laboratory in Winnipeg, Manitoba, for infection with West Nile virus (WNV). Using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), 558 mosquito pools (86% Culex species and 14% other species) had evidence of infection with WNV. Only 30% of the Culex specimens, however, were identified to the species level. In this study, Culex species-specific PCR primers were designed to identify individual mosquitoes and mixed pools of Culex mosquitoes to species. In addition, pools of non-Culex mosquitoes that tested positive for WNV were also screened for Culex DNA to determine the frequency of cross-contamination among mosquitoes of different species. All DNA extracts from 121 Culex and 51 non-Culex pools, previously positive for WNV, were screened, and Culex DNA was detected in approximately 6% of non-Culex pools. This study demonstrates that contamination among mosquito species can occur and emphasizes that precautions should be taken to minimize this potentially confounding effect.

Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae)

Nematology , 2010, Vol. 12(1), 157-160 Short communication Species-specific polymerase chain reaction primers for simple detection of Bursaphelenchus fraudulentus (Nematoda: Parasitaphelenchidae) Anna F I L I P I A K 1 , ∗ , Agata J AKUBOW SKA 1 , 2 and Marek T OMALAK 1 Bursaphelenchus fraudulentus Ruhm, 1956 (Nema- toda: Parasitaphelenchidae) is a tree-inhabiting species frequently found in Europe (Ruhm, 1956; Schauer-Blume & Sturhan, 1989; Tomalak, 2004; Carletti et al ., 2005) and Asia (Braasch et al ., 2001). It is primarily found in dy- ing or dead deciduous trees (Ruhm, 1956; Schauer-Blume & Sturhan, 1989), although occasionally reported from conifers (Braasch et al ., 2001). Within the genus the spe- cific morphological characters (i.e. , vulval flap, shape of spicules, position of caudal papillae and presence of four incisures in the lateral fields) place B. fraudulentus into a distinct xylophilus -group which comprises eight other, morphologically similar, species, including the quarantine pest B. xylophilus (Gu et al ., 2008). Reliable methods of taxonomic identification of these species are therefore of particular interest to plant quarantine services. Several molecular techniques have been developed and used for identification of Bursaphelenchus spp. (Wang et al. , 1999; Matsunaga & Togashi, 2004; Burgermeister et al ., 2005; Castagnone et al. , 2005; Leal et al ., 2005, 2007) with ITS-RLFP analysis the most widely used in research and quarantine practice. An alternative method, based on a simple PCR amplification with primers specific for B. xylophilus and B. mucronatus (Matsunaga & Togashi, 2004), enables rapid and precise species identification, even from a single nematode (Filipiak et al ., 2007). Both native European species from the xylophilus - group, i.e. , B. mucronatus and B. fraudulentus , are rela- tively common and harmless to trees. They can be mor- phologically difficult to distinguish from each other and from the quarantine pest B. xylophilus. The provision of PCR primers specific for the DNA of B. fraudulentus , in addition to those already existing for B. xylophilus and B. 1 Department of Biological Pest Control and Quarantine, Institute of Plant Protection, Wladyslawa Wegorka 20, Poznan, 60-318, Poland 2 Department of Genetics, University of Valencia, Dr. Moliner 50, 46-100, Burjassot, Spain ∗ Corresponding author, e-mail: A.Filipiak@ior.poznan.pl Received: 22 October 2008; revised: 26 June 2009 Accepted for publication: 29 June 2009 Keywords: Bursaphelenchus xylophilus -group, diagnostics, molecular. mucronatus (Matsunaga & Togashi, 2004), enhances the utility of this method. The main objectives of our research were to sequence the ITS regions of the B. fraudulentus genome and to design specific primers for PCR amplifica- tion. Eight isolates of B. fraudulentus , two of B. mucrona- tus and one of B. xylophilus were examined. The Aus- trian (Osterreich), Russian (DE10w), German (Helmstedt and H26), and Hungarian (Ungarn) isolates of B. fraudu- lentus and the isolate of B. xylophilus (China) were ob- tained from the nematode collection of the Federal Re- search Centre for Cultivated Plants (formerly Federal Bi- ological Research Centre for Agriculture and Forestry – BBA), Braunschweig, Germany (Dr T. Schroder). All the remaining nematode populations were isolated from in- fested trees in Poland. The populations of B. fraudulentus originated from Poznan (PL-01 and PL-05) and Kornik (PL-04) and those of B. mucronatus from Nowy Tomysl (NTo-01) and Slawa Slaska (SlS-01). Their taxonomic identification was originally based on morphological and morphometric characteristics and subsequently confirmed by ITS-RLFP analysis according to the protocol described by Burgermeister et al. (2005). Prior to examination, all isolates were reared on Botrytis cinerea /malt agar (4.5%) at 25 ◦ C for ca 2 weeks. Propagated nematodes were col- lected by the Baermann funnel method and stored in 10 μ l H 2 O at − 20 ◦ C until use. Extraction of DNA was performed according to the method described by Iwahori et al. (1998), with mi- nor modifications to the composition of the lysis buffer: 100 mM Tris, pH 8.5, 100 mM NaCl, 50 mM EDTA, 1% SDS, 1% β -mercaptoethanol, 100 μ g ml − 1 proteinase K per 100 μ l buffer. Before incubation, the mixture was © Koninklijke Brill NV, Leiden, 2010 DOI:10.1163/138855409X12469543767319 Also available online - www.brill.nl/nemy 157

Rapid Molecular Identification of Armored Scale Insects (Hemiptera: Diaspididae) on Mexican ‘Hass’ Avocado

'Hass' avocado, Persea americana Mill., fruit imported into California from Mexico are infested with high levels of armored scale insects (Hemiptera: Diaspididae), constituting several species. The paucity and delicate nature of morphological characters traditionally used to diagnose armored scales often require careful preparation of slide-mounted specimens and expert knowledge of the group, for their accurate identification. Here, we present a simple, quick, and accurate means to identify armored scales on Mexican avocados, based on amplification of the internal transcribed spacer two of ribosomal DNA, by using the polymerase chain reaction (PCR). This region seems to show a high level of intraspecific conformity among scale specimens originating from different localities. A suite of species-specific reverse PCR primers are combined in a single reaction, with a universal forward primer, and produce a PCR product of a unique size, that after standard gel electrophoresis, allows the direct diagnosis of six diaspidid species: Abgrallaspis aguacatae Evans, Watson & Miller; Hemiberlesia lataniae (Signoret); Hemiberlesia sp. near latania; Hemiberlesia rapax (Comstock); Acutaspis albopicta (Cockerell); and Pinnaspis strachani (Cooley). Two additional species, Diaspis miranda (Cockerell) and Diaspis sp. near miranda, also are separated from the others by using this method and are subsequently diagnosed by secondary digestion of the PCR product with the restriction endonuclease smaI.

Curly Top Survey in the Western United States

Curly top in sugar beet continues to be a challenging disease to control in the western United States. To aid in development of host resistance and management options, the curtovirus species composition was investigated by sampling 246 commercial fields along with nursery and field trials in the western United States. DNA was isolated from leaf samples and the species were identified using species-specific polymerase chain reaction primers for the C1 gene. Amplicons from 79 isolates were also sequenced to confirm identifications. Beet severe curly top virus (BSCTV) and Beet mild curly top virus (BMCTV) were widely distributed throughout the western United States, while only a few isolates of Beet curly top virus (BCTV) were found. In phylogenetic analysis, BSCTV, BMCTV, and BCTV isolates formed distinct groups in the dendrogram. Seven isolates not amplifiable with species-specific primers did amplify with curly top coat protein primers, indicating novel curtovirus species or strains may be present. Given the wide host range of the viruses responsible for curly top, frequent co-infections, and genetic diversity within and among species, establishing better host resistance, and controlling curly top will continue to be a challenge.

A NOVEL CLINICAL SYNDROME AND DETECTION OF ANAPLASMA OVIS IN MONGOLIAN REINDEER (RANGIFER TARANDUS)

The Tsaatan (or Dhuka) peoples of northern-western Mongolia are one of the few remaining reindeer-herding cultural groups in the world. Recently a disease condition that involves sudden death of reindeer and cases involving fever, lethargy, and pale mucous membranes has been reported. Examination of blood smears collected in the 2005 field season resulted in the identification of intra-erythrocytic inclusions resembling Anaplasma spp. in smears from clinically sick animals. Using universal polymerase chain reaction (PCR) primers for the amplification of the 60 kDa chaperonin gene (cpn60, also known as hsp60 or groEL), we detected sequences corresponding to Anaplasma ovis in reindeer blood samples. Species-specific PCR primers for A. ovis were designed and validated and used to screen blood samples from Mongolian reindeer. Screening of 66 blood samples collected in the 2006 field season resulted in the detection of A. ovis in 80% of the samples. Our results indicate a high prevalence of A. ovis in the Tsaatan reindeer herds and an association with clinical disease that is likely to be anaplasmosis. To our knowledge this is the first report of natural A. ovis infection in reindeer.

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the beta-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.

두 종의 사과 심식나방류 [복숭아순나방 (Grapholita molesta), 복숭아심식나방 (Carposina sasakii)] 동정용 DNA 분자지표

국내 서식하는 복숭아순나방 (Grapholita molesta (Busck))과 복숭아심식나방 (Carposina sasakii (Matsumura))의 유충은 사과 과실내부를 섭식하여 피해를 주는 해충이다. 사과를 수출할 때 복숭아심식나방은 수출대상국들로부터 검역 대상해충이다. 반면에 복숭아순나방은 광범위한 분포로 비교적 수입국으로부터 검역 대상 해충은 아니지만, 사과 과실 내부에서 발견되는 경우 복숭아심식나방으로 오인될 수 있다. 이는 발견되는 유충을 가지고 형태적으로 두 종을 구분하기 어렵기 때문이다. 특별히 수입국 검역단계에서 이러한 불완전한 동정 실태는 수출 사과의 폐기 또는 반송과 수출중단 등과 같은 막대한 경제적 손실을 초래하게 된다. 이에 이들을 구분할 수 있는 분자지표 개발이 요구되었다. 두 종의 미토콘드리아 DNA를 대상으로 다형을 보이는 여러 영역의 염기서열을 분석하였다. 이 서열을 바탕으로 진단용 제한위치가 결정되고 종 특이적 프라이머가 제작되었다. 본 연구는 세 부위의 종 특이적 제한효소 위치에 따라 PCR-RFLP 기술과 종 특이적 프라이머를 이용하여 진단용 PCR 기술을 개발하였다. Two fruit moths of the oriental fruit moth, Grapholita molesta (Busck), and the peach fruit moth, Carposina sasakii (Matsumura), infest apples in Korea by internally feeding behavior. C. sasakii is a quarantine insect pest from some other countries importing Korean apples. G. molesta is not a quarantine insect pest, but can be incorrectly identified as C. sasakii especially when it is found inside apple fruits at its larval stages because it is not easy to identify the two species by morphological characters alone. This incomplete identification results in massive economical loss by fruits needlessly destroyed or turned away at border inspection stations of the importing nations. This difficulty can be overcome by molecular DNA markers. Several polymorphic regions of mitochondrial DNA of both species were sequenced and used for developing specific striction sites and polymerase chain reaction (PCR) primers. Based on these sequences, three diagnostic PCR-restriction fragment length polymorphism (RFLP) sites were detected and validated for their practical uses. Also, species-specific PCR primers were devised to develop diagnostic PCR method for identifying the internal feeders.

우리나라에 분포하는 고추와 토마토 풋마름병균(Ralstonia solanacearum) 계통들의 유전적 다양성

풋마름병징을 나타내는 고추와 토마토 식물체로부터 35개의 풋마름병균 계통들을 분리하여 생리.생화학 실험, 병원성 실험, 유전학적 실험을 통해 유전적 다양성을 연구하였다. 생리 생화학 실험과 종특이적 polymerase chain reaction(PCR)을 실시한 결과 풋마름병균 계통들은 모두 Ralstonia solanacerum biovar 4로 동정되었다. 분리균은 고추와 토마토 유묘를 이용해 병원성이 확인되었다. Repetitive sequence-based PCR(rep-PCR) 결과를 토대로 계통도 분석을 한 결과 고추와 토마토 분리균은 6개의 group으로 나뉘었으며, 유전적 다양성은 높게 나타났다. 풋마름병균 계통들의 그룹간에는 지역별, 기주별 특이성은 관찰되지 않았다. A total of 35 strains of Ralstonia solanacearum isolated from wilted pepper and tomato plants in Korea were analyzed for their genetic diversity by bacteriological, pathological and molecular biological approaches. All the strains were identified as R. solanacearum biovar 4 on the basis of physiological and biochemical tests, and species-specific PCR primers. Pathogenicity of the strains was confirmed by inoculating on 4-week-old pepper and tomato seedlings. Using cluster analysis based on repetitive sequence-based polymerase chain reaction (rep-PCR) genomic fingerprints, R. solanacearum strains isolated from pepper and tomato in Korea divided into 6 groups showing a high degree of genetic diversity at 55% similarity level. The genetic diversify of strains was not significantly correlated with their geographic origins and host plants.