Three pairs of specific primers were developed for rapid and precise RT-PCR detection of three seed-transmissible viruses, namely Peanut clump virus (PCV, Pecluvirus), White clover mosaic virus (WCIMV, Potexvirus) and Carrot red leaf virus (CaRLV, Luteovirus). Each primer set was found in conserved ...
Three pairs of specific primers were developed for rapid and precise RT-PCR detection of three seed-transmissible viruses, namely Peanut clump virus (PCV, Pecluvirus), White clover mosaic virus (WCIMV, Potexvirus) and Carrot red leaf virus (CaRLV, Luteovirus). Each primer set was found in conserved region through multiple sequence alignment in the DNAMAN. Total nucleic acids extracted from PCV-, WCMV-, and CaRLV-infected seeds and healthy plants were used for RT-PCR detection using each virus-specific primer, Sizes of PCV, WCIMV, and CaRLV PCR products were 617bp (PCV-uni5 and PCV-uni3 primers), 561bp (WCMV-CP5 and WCMV-CP3 primers), and 626bp (CL1-UP and CL2-DN primers); which corresponded to the target sizes. Nucleotides sequences of each amplified cDNA were confirmed which belonged to the original virus. This study suggests that these virus-specific primer sets can specifically amplify viral sequences in infected seeds. Thus, they can be used for specific detection of three viruses (PCV, WCMV and CaRLV) from imported seed samples for plant quarantine service.
Three pairs of specific primers were developed for rapid and precise RT-PCR detection of three seed-transmissible viruses, namely Peanut clump virus (PCV, Pecluvirus), White clover mosaic virus (WCIMV, Potexvirus) and Carrot red leaf virus (CaRLV, Luteovirus). Each primer set was found in conserved region through multiple sequence alignment in the DNAMAN. Total nucleic acids extracted from PCV-, WCMV-, and CaRLV-infected seeds and healthy plants were used for RT-PCR detection using each virus-specific primer, Sizes of PCV, WCIMV, and CaRLV PCR products were 617bp (PCV-uni5 and PCV-uni3 primers), 561bp (WCMV-CP5 and WCMV-CP3 primers), and 626bp (CL1-UP and CL2-DN primers); which corresponded to the target sizes. Nucleotides sequences of each amplified cDNA were confirmed which belonged to the original virus. This study suggests that these virus-specific primer sets can specifically amplify viral sequences in infected seeds. Thus, they can be used for specific detection of three viruses (PCV, WCMV and CaRLV) from imported seed samples for plant quarantine service.
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문제 정의
Therefore, the purpose of this study is to establish a rapid and exact detection technique for these viruses. For this objective, we used RT-PCR technique and this powerfully detected specific target virus from quite a few viruses into seeds.
This study showed a useful model test with effective detection method of seed-transmissible viruses in plant quarantine.
가설 설정
Besides, information on virus genome sequence amplified specific region of these viruses. In this study, we suggested proper total RNA extraction and RT-PCR condition for these three viruses and these conditions were applied to quarantine of three viruses.
제안 방법
Moreover, designed primer of three pairs was confirmed to have equal yield under ±5℃ of annealing temperature and exact amplification of target size. Although total RNAs of seeds were not detected by RT-PCR, this procedure was able to detect PCY WCMY and CaRLV from infected seed samples.
CaRLV-infected tissues were kindly supplied from Bejo Seed Company in the Netherlands. These viruses were officially imported with clearance from the National Plant Quarantine Service (NPQS), and all experiments were conducted under close collaboration with NPQS staff Total RNA extraction. Total RNAs were extracted from PCV-, WCIMV-, and CaRLV-infected tissues.
대상 데이터
Oligonucleotides primers design. Degenerated or specific primers were obtained from conserved regions of PCV and WC1MV sequences from the GenBank of the National Center for Biotechnology Information (PCV: L07269, X76658, AH009203, AF239731, AF239729, AF447369, AF447399, AF447400, AF447401, NC_003668; WCIMV: NC_003820, XI6636, Ml 8920). In the case of CaRLY a pair of degenerated primer was designed by and taken from Dr.
이론/모형
Total RNAs were extracted from PCV-, WCIMV-, and CaRLV-infected tissues. All genomic RNAs were extracted with SDS-proteinase K/phenol method as described by Ryu et al. (1995) and Rneasy Plant Mini Kit (Qiagen, USA) according to the manufacturer's protocol (Table 1). Each sample of freeze-dry leaf tissues was ground in extraction buffer (50 mM Tris (pH 8.
성능/효과
Lane M: 1 kb plus DNA ladder, Lane 1: PCV total RNA. (B) Electrophoretic pattern of amplified products of conserved region of WCMV-infected leaf tissues with WC1MV-CP5/3 primer set Lane M: 1 kb plus DNA ladder, Lane 1: WCMV total RNA (C) Electrophoretic pattern of amplified products of conserved region of CaRLV-infected leaf tissues with CL1UP/2DN Lane M: 1 kb plus DNA ladder, Lane 1: CaRLV total RNA.
참고문헌 (17)
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