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Development of Nested PCR Primer Set for the Specific and Highly Sensitive Detection of Human Parvovirus B19
Biomed Sci Lett 2018;24:390-397
Published online December 31, 2018;  https://doi.org/10.15616/BSL.2018.24.4.390
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Kyu-Bong Cho†,*

Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu 11644, Korea
Correspondence to: Kyu-Bong Cho. Department of Biomedical Laboratory Science, Shinhan University, 95, Hoam-ro, Uijeongbu-si, Gyeonggi-do 11644, Korea. Tel: +82-31-870-3712, Fax: +82-31-870-3719, e-mail: kbcho@shinhan.ac.kr
Received November 19, 2018; Revised December 8, 2018; Accepted December 13, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract

For the specific detection of human Parvovirus B19 (HuPaV-B19), we designed ten specific PCR primers from 3,800~4,500 nucleotides of HuPaV-B19 complete genome (NC_000883.2). Seventeen candidate PCR primer sets for specific detecting HuPaV-B19 were constructed. In specific reaction of HuPaV-B19, seventeen PCR primer sets showed specific band, however five PCR primer sets were selected basis of band intensity, amplicon size and location. In nonspecific reaction with seven reference viruses, four PCR primer sets showed non-specific band, however one PCR primer set is not. Detection sensitivity of final selective PCR primer set was 100 fg/μL for 112 minute, and PCR amplicon is 539 base pairs (bp). In addition, nested PCR primer set was developed, for detection HuPaV-B19 from a low concentration of contaminated samples. Selection of nested PCR primer set was basis of sensitivity and groundwater sample tests. Detection sensitivity of final selective PCR and nested PCR primer sets for the detection of HuPaV-B19 were 100 fg/μL and 100 ag/μL basis of HuPaV-B19 plasmid, it was able to rapid and highly sensitive detection of HuPaV-B19 than previous reports. We expect developed PCR primer set in this study will used for detection of HuPaV-B19 in various samples.

Keywords : Human Parvovirus B19, Polymerase chain reaction, Nested PCR
꽌濡

궗엺 뙆蹂대컮씠윭뒪 B19 (human Parvovirus B19; HuPaVB19)뒗 group II ssDNA virus, Parvoviridae, Erythroparvovirus濡 遺꾨쪟릺硫(Heegaard and Brown, 2002), 냼븘 쟾뿼꽦 솉諛 諛 꽦씤 瑜섎쭏떚뒪꽦 愿젅뿼 벑쓽 利앹긽씠 굹궃떎(White et al., 1985). 씪諛섏쟻쑝濡쒕뒗 샇씉湲곕 넻빐 媛먯뿼릺뒗 寃껋쑝濡 븣젮졇 엳뿀쑝굹, 닔삁, 옣湲 씠떇 벑 엫긽쟻 媛먯뿼 궗濡肉먮쭔 븘땲씪 쓬슜닔뿉꽌 삤뿼씠 蹂닿퀬맂 諛 엳떎(Casteel et al., 2002; Schmidt et al., 2007; Marano et al., 2015). 理쒓렐 HuPaV-B19瑜 吏꾨떒븯湲 쐞븳 諛⑸쾿쑝濡 以묓빀슚냼뿰뇙諛섏쓳(polymerase chain reaction; PCR)쓣 湲곕컲쑝濡 븳 듅씠쟻 빑궛 떒렪 利앺룺씠 솢슜릺怨 엳떎(Bonvicini et al., 2009; Bock et al., 2014). PCR 諛⑸쾿 以 conventional PCR 留롮 뿰援щ줈 븞젙꽦씠 寃利앸릺뿀怨, 遺꾩꽍옄쓽 꼻 솢슜痢듭쓣 媛吏怨 엳쑝硫, 엫긽 諛 鍮 엫긽 떆猷뚯뿉꽌 솢슜븷 닔 엳뼱 몴以솕 諛⑸쾿쑝濡 쟻빀븯떎(Park et al., 2016; Cho, 2018). 洹몃윭굹 떎닔쓽 뿰援ъ옄뱾뿉 쓽빐 HuPaV-B19瑜 吏꾨떒븯뒗 conventional PCR primer 議고빀씠 蹂닿퀬(Sevall et al., 1992; Bergallo et al., 2008; Bonvicini et al., 2009; Bock et al., 2014)릺怨 엳怨, 듅씠꽦, 寃異 誘쇨컧룄, 諛섏쓳 떆媛, 鍮꾩슜 벑쓽 鍮꾧탳 뿰援ш 誘명씉븯떎. 븳렪, 쓬슜닔뿉꽌 HuPaV-B19媛 寃異쒖뿉 뵲씪 엫긽 쇅 떆猷뚯뿉꽌룄 紐⑤땲꽣留곸씠 븘슂븯떎. 洹몃윭굹 쓬슜닔 벑 臾쇳솚寃쎌 엫긽 떆猷뚮뱾뿉 鍮꾪빐 긽쟻쑝濡 諛붿씠윭뒪媛 誘몃웾 삤뿼맆 媛뒫꽦씠 겕湲 븣臾몄뿉 넂 寃異 誘쇨컧룄媛 슂援щ맂떎(Cho, 2018). 삉븳 엫긽怨 鍮 엫긽 벑 떆猷 蹂꾨줈 솢슜븯뒗 PCR 議곌굔 諛 HuPaV-B19 빑궛씠 利앺룺릺뒗 쐞移섍 떖씪꽌 쑀쟾삎 뿰援, 떆猷 蹂 뿰怨 뿰援 벑뿉 뼱젮씠 뵲瑜몃떎. 뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 HuPaV-B19 듅씠쟻 吏꾨떒씠 媛뒫븳 PCR primer 議고빀 諛 넂 寃異 誘쇨컧룄濡 寃異쒗븷 닔 엳뒗 nested PCR primer 議고빀쓣 媛쒕컻븯쑝硫, 湲곗〈뿉 蹂닿퀬맂 HuPaV-B19 conventional PCR primer 議고빀뱾怨 鍮꾧탳븯떎. 삉븳 理쒖쥌 꽑諛쒗븳 PCR 諛 nested PCR primer 議고빀쓽 떆猷 쟻슜꽦 뿰援щ 닔뻾븯떎.

옱猷 諛 諛⑸쾿

PCR primer 꽕怨

誘멸뎅 援由쎌깮臾쇱젙蹂댁꽱꽣뿉꽌 HuPaV-B19 complete genome(NC_000883.2) 뿼湲곗꽌뿴쓣 닔吏묓븯쑝硫, 씠 以 援ъ“ 떒諛깆쭏쓣 肄붾뵫븯뒗 HuPaV-B19 VP1 VP2 gene쓣 긽쑝濡 븯떎. DNAMAN software package version 6.0 (Lynnon Biosoft, Quebec, Canada)濡 듅씠쟻 primer 꽕怨 썑 Oligo Calculator version 3.27濡 potential hairpin formation 벑쓽 뿬遺瑜 솗씤븯쑝硫(Kibbe, 2007), 珥 10媛쒖쓽 primer뱾쓣 젣옉븯떎(Fig. 1).

Fig. 1.

Map of specific PCR primers for the detection of human Parvovius B19.



諛붿씠윭뒪 빑궛 닔吏

HuPaV-B19 (NC_000883.2)쓽 VP1 諛 VP2 援ш컙 2,000 nucleotides (3,001~5,000)瑜 빀꽦븯뿬 plasmid솕 븯쑝硫, 鍮 듅씠쟻 떎뿕뿉 솢슜븳 7醫낆쓽 李멸퀬 諛붿씠윭뒪(human enteric Adenovirus-41, Aichivirus A, human Astorivirus, Parechovirus A, PoliovirusSapovirus)쓽 빑궛쓣 닔吏묓븯떎.

PCR 諛 nested PCR primer 꽑諛

HuPaV-B19 듅씠쟻 primer 10媛쒕 湲곗큹濡 PCR 利앺룺씠 媛뒫븳 17媛 PCR 議고빀쓣 援ъ꽦븯떎(Table 1). 援ъ꽦븳 議고빀뿉꽌 HuPaV-B19 빑궛뿉 듅씠쟻 諛 李멸퀬 諛붿씠윭뒪뿉 鍮꾪듅씠쟻 諛섏쓳쓣 寃젙븯떎. 듅씠쟻 諛섏쓳留 굹궃 PCR primer 議고빀뱾쓽 寃異 誘쇨컧룄 遺꾩꽍 HuPaV-B19 plasmid瑜 1 ng/μL瑜 썝븸쑝濡 10-8 (10 ag/μL)源뚯 10諛 떒怨 씗꽍븯뿬 諛섏쓳쓣 遺꾩꽍 썑 理쒖쥌 븯굹쓽 議고빀쓣 꽑諛쒗븯떎. 삉븳 寃異 誘쇨컧룄 떆뿕쓣 넻빐 nested PCR 1媛쒖쓽 primer 議고빀쓣 꽑諛쒗븯떎. PCR 議곗꽦 AccuPower® HotStart PCR PreMix (Bioneer, Korea), 젙諛⑺뼢 諛 뿭諛⑺뼢 봽씪씠癒 (25 pmol) 媛 1 μL (珥 2 μL), 二쇳삎 빑궛 1 μL 諛 nucleic acid free water 17 μL濡 理쒖쥌 20 μL濡 븯쑝硫, 議곌굔 95°C뿉꽌 10遺꾧컙 蹂꽦 썑, 35쉶 諛섎났(95°C 45珥, 55°C 1遺 諛 72°C 1遺) 썑 72°C 5遺 媛 諛섏쓳븯떎. PCR 궛臾쇱 1.2% agarose gel뿉 濡쒕뵫 썑 100 V뿉꽌 30遺 룞븞 쟾湲곗쁺룞 븯쑝硫, UV illuminator뿉꽌 寃곌낵瑜 솗씤븯떎.

PCR and nested PCR primer sets for the detection of HuPaV-B19

 Primer set  PCR type  Primer name  Sequences (5’ → 3’) Amplicon size (bp) Sequence location Reference
1~17 PCR and nested PCR -* 323~834 3,280~4,229 This study

Ref. 01 PCR TJI TTCTTTTCAGCTTTTAGG 200 3,775~3,792 [8]
TJII GTACTTCTGGTACGTTAAGT 3,956~3,975

Nested PCR 968 TATAAGTTTCCTCCAGTGCC 120 3,818~3,837
967b TGTAATCCTCCACTGGGTT 3,920~3,938

Ref. 02 PCR F1 TATGCTTACTTAACAGTAGG 852 3,566~3,585 4,417~4,400 [14]
R1 AATTGGCCCACTTTGTGG

Nested PCR F2 GCCTTAGCACAGGTACCTCT 397 3,960~3,979
R2 CATCCTCCTAAGGCTGCAAAC 4,234~4,253

Ref. 03 PCR 1stForward CAAAAGCATGTGGAGTGAGG 398 3,187~3,206 3,584~3,565 [7]
1stReverse CTACTAACATGCATAGGCGC

Nested PCR 2ndForward CCCAGAGCACCATTATAAGG 288 3,271~3,290
2ndReverse GTGCTGTCAGTAACCTGTAC 3,558~3,539

Ref. 04 PCR Vp2i-Forward CTAGAATATCCTTACGCCCTGG 386 4,274~4,295 4,660~4,640 [16]
Vp2i-Reverse GTGGCTGATGCAAACCCCATC

Nested PCR Vp2p-Forward CCATTTCTCATGGTCAGACCAC 193 4,355~4,376
Vp2p-Reverse CCATACAGAACCCACCATTAGG 4,548~4,527

Ref. 05 PCR Primer A AGCATGACTTCAGTTAATTC 727 3,122~3,141 3,868~3,887 [17]
Primer B GATTGTACATTTCATAAAAG

Ref. 06 PCR 1STForward AGCATGTGGAGTGAGGGGGC 290 No Data [9]
1STReverse AAAGCATCAGGAGCTATACTTCC

Nested PCR 2NDForward GCTAACTCTGTAACTTGTAC 173
2NDReverse AAATATCTCCATGGGGTTGAG

Sequences of final selective PCR primer were shown in Table 3



湲곗〈 떎뿕 鍮꾧탳

쁽옱源뚯 蹂닿퀬맂 HuPaV-B19 吏꾨떒슜 conventional PCR primer 議고빀(Yamakawa et al., 1995; Cassinotti et al., 1993; Bergallo et al., 2008; Bock et al., 2014)뱾怨 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 諛⑸쾿쓽 寃異 誘쇨컧룄, 諛섏쓳 떆媛, 떆猷 쟻슜꽦 벑쓣 鍮꾧탳븯떎(Table 1). 寃異 誘쇨컧룄 鍮꾧탳뒗 HuPaV-B19 plasmid 떒怨 씗꽍븸쓣 궗슜븯怨, 諛섏쓳 떆媛꾩 PCR씠 諛섏쓳븯뒗뜲 嫄몃━뒗 떆媛꾩쑝濡 븯떎.

떆猷 쟻슜꽦 떆뿕

떆猷 쟻슜꽦 遺꾩꽍쓣 쐞븯뿬 20媛쒖쓽 吏븯닔 떆猷뚮 엫쓽濡 梨꾩랬븯쑝硫, 깉由 諛 냽異 썑 DNeasy® PowerWater® Kit (Qiagen, Germany)濡 珥 빑궛쓣 異붿텧븯떎. 蹂 뿰援ъ뿉꽌 媛쒕컻븳 PCR 諛 nested PCR primer 議고빀쓣 솢슜븯뿬 吏븯닔뿉꽌 異붿텧븳 珥 빑궛쓣 二쇳삎쑝濡 HuPaV-B19瑜 吏꾨떒븯떎.

寃곌낵

PCR 諛 nested PCR primer 꽑諛

HuPaV-B19 듅씠쟻 PCR primer 17媛 議고빀뿉꽌 紐⑤몢 듅씠쟻 諛대뱶媛 굹궗떎. 씠 以 諛대뱶쓽 利앺룺 젙룄, 겕湲, 쐞移 벑쓣 怨좊젮븯뿬 5媛쒖쓽 PCR primer 議고빀(#03, #06, #10, #13 諛 #16)쓣 꽑諛쒗븯떎(Fig. 2). 꽑諛쒗븳 PCR primer 議고빀 5媛 以 4媛(#06, #10, #13 諛 #16) 議고빀뿉꽌 李멸퀬 諛붿씠윭뒪 빑궛뿉 鍮 듅씠쟻 諛섏쓳씠 굹궗쑝硫, 1媛(#03) 議고빀 鍮꾪듅씠쟻 諛섏쓳씠 굹굹吏 븡븯떎(Fig. 3). PCR primer 議고빀 #03 539 base pairs (bp) 겕湲곗쓽 HuPaV-B19 빑궛 떒렪쓣 듅씠쟻쑝濡 利앺룺븯쑝硫, 寃異 媛먮룄뒗 100 fg/μL源뚯濡 遺꾩꽍릺뿀떎(Fig. 4). PCR primer 議고빀 #03씠 利앺룺븳 빑궛 떒렪 539 bp뿉꽌 利앺룺씠 媛뒫븳 3媛쒖쓽 ested PCR primer 議고빀쓣 꽕怨꾪븯떎. 寃異 誘쇨컧룄 遺꾩꽍 寃곌낵 1 fg/μL~100 ag/μL 닔以쑝濡 HuPaV-B19 듅씠쟻 빑궛 떒렪쓣 利앺룺븷 닔 엳뿀쑝硫, 씠 以 넂 寃異 誘쇨컧룄媛 굹궃 2媛쒖쓽 썑蹂 nested PCR primer 議고빀쓣 꽑諛쒗븯떎(Fig. 4).

Fig. 2.

Specific reaction of seventeen PCR primer sets for detection of HuPaV-B19. M, 100 bp Ladder marker (MGMed, Korea); lane number, number of PCR primer set (#01~17); N, negative control; dot, selective PCR primer set.


Fig. 3.

Non-specific reaction of five primer sets basis of seven refernece viruses. Lane M, 100 bp Ladder marker; P, positive control; N, negative control; AdV41, human Adenovirus 41; Ai, Aichivirus; Ast, Astrovirus; Pe, Parechovirus A; Po, Poliovirus; S, Sapovirus; N, Norovirus GII; dot, selective PCR primer sets.


Fig. 4.

Sensitivity of PCR and candidate nested PCR primer sets. Lane M, 100 bp Ladder marker; lane number, dilution rates; N, negative control; PN, PCR negative control.



湲곗〈 議곌굔 鍮꾧탳

HuPaV-B19 寃異쒖슜 conventional PCR primer 議고빀 6媛쒕 긽쑝濡 寃異 誘쇨컧룄瑜 遺꾩꽍븳 寃곌낵 븳 踰덉쓽 PCR뿉꽌뒗 1 ng/μL 씠븯뿉꽌 100 fg/μL 닔以씠뿀쑝硫, nested PCR 寃곌낵 1 ng/μL뿉꽌 10 fg/μL 닔以쑝濡 굹궗떎(Table 2). 븳 踰덉쓽 PCR뿉꽌 寃異 誘쇨컧룄媛 넂븯뜕 議고빀 鍮꾧탳 primer 5踰덉쑝濡 諛섏쓳 떆媛꾩씠 130遺꾩씠뿀쑝硫, nested PCR 寃異 誘쇨컧룄媛 媛옣 넂븯뜕 鍮꾧탳 primer 議고빀 3踰덇낵 6踰덉쑝濡 PCR 떆媛꾩쓣 룷븿븯뿬 媛곴컖 380遺꾧낵 136遺꾩씠뿀떎. 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 PCR primer 議고빀 鍮꾧탳 primer 5踰덇낵 룞씪 닔以쓽 寃異 誘쇨컧룄媛 굹궗쑝굹 諛섏쓳 떆媛꾩쓣 빟 18遺 떒異뺥븯떎. 삉븳 nested PCR primer 議고빀 諛섏쓳 떆媛꾩씠 珥 224遺꾩쑝濡 鍮꾧탳 primer 6踰덉뿉 鍮꾪빐 88遺 諛섏쓳 떆媛꾩씠 湲몄뿀쑝굹 빟 100諛 씠긽쓽 寃異 誘쇨컧룄媛 굹궗떎(Table 2).

Comparison of performance between this study and previously reported PCR primer sets

Primer set Reaction time (min)  Sensitivity
This study PCR 112 100 femto gram
Nested PCR* 224 100 atto gram

Ref. primer set 1 PCR 220 > 1 nano gram
Nested PCR* 440 10 pico gram

Ref. primer set 2 PCR 86 10 pico gram
Nested PCR* 131 100 femto gram

Ref. primer set 3 PCR 190 100 pico gram
Nested PCR* 380 10 femto gram

Ref. primer set 4 PCR 130 1 pico gram
Nested PCR* 220 1 pico gram

Ref. primer set 5 PCR 230 1 pico gram

Ref. primer set 6 PCR 68 100 pico gram
Nested PCR* 136 10 femto gram

Reaction time of nested PCR is the sum of PCR and nested PCR reaction times



떆猷 쟻슜꽦 떆뿕

蹂 뿰援ъ뿉꽌 媛쒕컻븳 PCR primer 議고빀 諛 썑蹂 nested PCR primer 議고빀 2媛쒕 긽쑝濡 떆猷 쟻슜꽦 떆뿕쓣 닔뻾븯떎. PCR 닔以뿉꽌뒗 20媛 떆猷 紐⑤몢 HuPaV-B19 쓬꽦쑝濡 굹궗쑝굹, nested PCR primer 議고빀 以 理쒖쥌 궛臾 464 bp瑜 利앺룺븯뒗 議고빀뿉꽌 HuPaV-B19 듅씠쟻 諛대뱶媛 굹굹(Fig. 5) 理쒖쥌 nested PCR primer 議고빀쑝濡 꽑諛쒗븯떎. 理쒖쥌 꽑諛쒗븳 PCR 諛 nested PCR primer 議고빀쓽 젙蹂대뒗 Table 3怨 媛숇떎.

Information of finally selective PCR and nested PCR primer set for the detection of HuPaV-B19

 PCR tpye   Primer   Sequence (5’ → 3’) Mer (nt) Amplicon size (bp)
PCR HuPaV-B19_F10 CAACGCCTCAGAAAAATACC 20 19 539
HuPaV-B19_R40 GGGCTAAAGTATCTTGACC

Nested PCR HuPaV-B19_F10 CAACGCCTCAGAAAAATACC 20 22 464
HuPaV-B19_R10 CTCCAGTATTAGAGCTGTCTCC

Fig. 5.

Test of twenty groundwater samples using the final selective PCR primer set and candidate two nested PCR primer sets. Lane M, 100 bp Ladder marker; Lane 1~20, groundwater sample; N, negative control; PN, PCR negative control; dot, selective nested PCR primer sets.


怨좎같

蹂 뿰援ъ뿉꽌뒗 HuPaV-B19 吏꾨떒슜 PCR 諛 nested PCR primer 議고빀쓣 媛쒕컻븯떎. HuPaV-B19瑜 吏꾨떒븷 닔 엳뒗 遺꾩꽍踰뺤 PCR 諛⑸쾿 쇅 슚냼硫댁뿭遺꾩꽍踰, 떣釉붾옃샎꽦솕踰 벑씠 솢슜맆 닔 엳떎(Schwarz et al., 1988; Mori et al., 1989; Brown et al., 1990; Gray et al., 1993; Bostic et al., 1999). 洹몃윭굹 듅씠꽦, 寃異 誘쇨컧룄, 遺꾩꽍 궃씠룄, 寃궗옄쓽 쐞뿕꽦, 鍮꾩슜 벑쓣 醫낇빀쟻쑝濡 怨좊젮븯뿬 빑궛 떒렪쓣 利앺룺븯뒗 湲곗닠씠 利앷븯怨 엳뒗 異붿꽭씠떎(Cho, 2018). 빑궛 떒렪 利앺룺 湲곕컲쓽 諛⑸쾿 以 real-time qPCR 듅씠꽦怨 寃異 誘쇨컧룄媛 넂怨 떊냽븯寃 吏꾨떒씠 媛뒫븯뿬 留롮 솢슜씠 蹂닿퀬(Sewell, 1995)릺怨 엳쑝굹 뿼湲곗꽌뿴 遺꾩꽍쓣 븷 닔 뾾뼱 쑀쟾삎 遺꾩꽍씠 뼱졄怨, 寃곌낵 遺꾩꽍 벑 conventional PCR뿉 鍮꾪빐 寃궗옄쓽 닕젴룄媛 븘슂븳 떒젏씠 엳떎. 삉븳 벑삩利앺룺踰(loop-mediated isothermal amplification) 鍮 듅씠쟻 쐞뼇꽦 諛섏쓳씠 留롮씠 굹굹怨 鍮꾩슜씠 鍮꾩떬 떒젏씠 蹂닿퀬릺怨 엳떎(Smith and Osborn, 2009). 씠뿉 鍮꾪빐 conventional PCR 뿼湲곗꽌뿴 遺꾩꽍씠 媛뒫븯怨 쐞 뼇꽦 諛섏쓳씠 긽쟻쑝濡 궙寃 굹굹誘濡 몴以 遺꾩꽍湲곗닠濡 쟻빀븯떎怨 궗猷뚮맂떎.

븳렪, 쓬슜닔 벑뿉꽌룄 HuPaV-B19媛 寃異쒕릺硫댁꽌(Casteel et al., 2002), 뼢 썑 엫긽 쇅 쓬슜닔瑜 룷븿븳 떇뭹, 뼱뙣瑜, 솚寃 떆猷 벑뿉꽌룄 HuPaV-B19 紐⑤땲꽣留곸씠 븘슂븷 寃껋쑝濡 蹂댁씤떎. 理쒓렐 援궡 踰붾泥 닔씤꽦 · 떇뭹留ㅺ컻 諛붿씠윭뒪 삊쓽泥댁뿉꽌 Norovirus GI怨 GII瑜 寃궗븯뒗 몴以솕 諛⑸쾿쑝濡 conventional PCR쓣 梨꾪깮 諛 異붿쭊븯怨 엳떎(Park et al., 2016). 씠뿉 뵲씪 떎뼇븳 떆猷뚯뿉꽌 蹂묒썝泥댁쓽 寃궗瑜 닔뻾빐빞 븯뒗 寃궗踰 媛쒕컻 뿰援ъ쓽 뿰愿꽦, 寃궗옄쓽 렪由ъ꽦 벑씠 怨좊젮릺뼱빞 븳떎. 듅엳 臾 솚寃쎄낵 媛숈씠 諛붿씠윭뒪쓽 洹밸몃웾 삤뿼씠 삁긽릺뒗 떆猷뚯뿉꽌뒗 nested PCR 씠긽쓽 寃異 媛먮룄媛 븘슂븯떎(Kittigul et al., 2005; Cho, 2018). 씠踰 뿰援ъ뿉꽌 媛쒕컻븳 PCR primer 議고빀 빟 112遺 諛섏쓳쑝濡 HuPaV-B19瑜 듅씠쟻쑝濡 利앺룺븯쑝硫 궛臾쇱 539 bp떎. PCR primer 議고빀 湲곗〈뿉 蹂닿퀬맂 6媛쒖쓽 議고빀 以 媛옣 寃異 誘쇨컧룄媛 넂 議고빀怨 룞벑 닔以씠뿀쑝굹 諛섏쓳 떆媛꾩쓣 빟 18遺 떒異뺤떆耳곕떎. PCR primer 議고빀 怨 냽룄 媛먯뿼 삉뒗 삤뿼씠 삁긽릺뒗 엫긽, 떇뭹, 뼱뙣瑜 벑쓽 떆猷 寃궗뿉 쟻빀븷 寃껋쑝濡 깮媛곷맂떎. 삉븳 nested PCR primer 議고빀 빟 112遺꾩쓽 異붽 諛섏쓳쑝濡 100 ag/μL 닔以源뚯 寃異쒗븷 닔 엳뿀떎. 씠寃껋 湲곗〈뿉 蹂닿퀬맂 PCR primer 議고빀뱾 蹂대떎 寃異 誘쇨컧룄瑜 빟 100諛 닔以쑝濡 뼢긽떆궗 닔 엳뿀쑝硫, 吏븯닔 떆猷뚯뿉꽌룄 吏꾨떒씠 媛뒫븳 寃껋씠 솗씤릺뿀떎. Nested PCR primer 議고빀 誘몃웾 삤뿼씠 삁긽릺뒗 솚寃 떆猷뚯뿉꽌쓽 솢슜씠 쟻빀 븷 寃껋쑝濡 蹂댁씤떎.

蹂 뿰援ъ뿉꽌 媛쒕컻븳 PCR 諛 nested PCR primer 議고빀 湲곗〈 뿰援ъ뿉 鍮꾪빐 鍮좊Ⅸ 諛섏쓳 諛 넂 寃異 誘쇨컧룄濡 HuPaV-B19瑜 寃異쒗븷 닔 엳뿀떎. 씠踰 뿰援щ뒗 뼢썑 엫긽 諛 鍮 엫긽 떆猷 벑뿉꽌 HuPaV-B19瑜 吏꾨떒븯뒗 諛⑸쾿쑝濡 솢슜븷 닔 엳쓣 寃껋쑝濡 湲곕맂떎.

ACKNOWLEDGEMENT

蹂 끉臾몄 2018뀈룄 떊븳븰援 븰닠뿰援щ퉬 吏썝쑝濡 뿰援щ릺뿀쓬.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

References
  1. Bergallo M, Costa C, Sidoti F, Novelli M, Ponti R, and Castagnoli C et al. Variants of Parvovirus B19: bioinformatical evaluation of nested PCR assays. Intervirology 2008;51:75-80.
    Pubmed CrossRef
  2. Bock CT, Duchting A, Utta F, Brunner E, Sy BT, and Klingel K et al. Molecular phenotypes of human parvovirus B19 in patients with myocarditis. World J Cardiol 2014;26:183-195.
    Pubmed KoreaMed CrossRef
  3. Bonvicini F, Manaresi E, Gallinella G, Gentilomi GA, Musiani M, and Zerbini M. Diagnosis of fetal parvovirus B19 infection: value of virological assays in fetal specimens. BJOG 2009;116:813-817.
    Pubmed CrossRef
  4. Bostic JR, Brown KE, Young NS, and Koenig S. Quantitative analysis of neutralizing immune responses to human parvovirus B19 using a novel reverse transcriptase-polymerase chain reactionbased assay. J Infect Dis 1999;179:619-626.
    Pubmed CrossRef
  5. Brown CS, van Bussel MJ, Wassenaar AL, van Elsacker-Niele AM, Weiland HT, and Salimans MM. An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen. J Virol Methods 1990;29:53-62.
    Pubmed CrossRef
  6. Cassinotti P, Weitz M, and Siegl G. Human Parvovirus B19 infections: routine diagnosis by a new nested polymerase chain reaction assay. J Med Virol 1993;40:228-234.
    Pubmed CrossRef
  7. Casteel MJ, Gold A, Sobsey MD, and Brecher ME. Photosensitization of hepatitis A virus and other non-enveloped RNA viruses by mesosubstituted porphyrins in water and human plasma. Biologic Effects of Light 2001, Holick M.F. Boston, MA; 2002.
  8. Cho KB. Construction of Improved PCR Primer Set for the Detection of Human Enteric Adenovirus 41. Biomedical Science Letters 2018;24:230-238.
    CrossRef
  9. Gray JJ, Cohen BJ, and Desselberger U. Detection of human parvovirus B19-specific IgM and IgG antibodies using a recombinant viral VP1 antigen expressed in insect cells and estimation of time of infection by testing for antibody avidity. J Virol Methods 1993;44:11-23.
    Pubmed CrossRef
  10. Heegaard ED, and Brown KE. Human parvovirus B19. Clin Microbiol Rev 2002;15:485-505.
    Pubmed KoreaMed CrossRef
  11. Kibbe WA. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 2007;35:43-46.
    Pubmed KoreaMed CrossRef
  12. Kittigul L, Ekchaloemkiet S, Utrarachkij F, Siripanichgon K, Sujirarat D, and Pungchitton S et al. An efficient virus concentration method and RT-nested PCR for detection of rotaviruses in environmental water samples. J Virol Methods 2005;124:117-122.
    Pubmed CrossRef
  13. Marano G, Vaglio S, Pupella S, Facco G, Callizzani G, and Candura F et al. Human parvovirus B19 and blood product safety: a tale of twenty years of improvements. Blood Transfus 2015;13:184-186.
    Pubmed KoreaMed CrossRef
  14. Mori J, Field AM, Clewley JP, and Cohen BJ. Dot blot hybridization assay of B19 virus DNA in clinical specimens. J Clin Microbiol 1989;27:459-464.
    Pubmed KoreaMed
  15. Park S, Lee S, Chung H, Park J, and Park S et al. Development and verification of genetically diagnostic method for the detection of non-regulated viruses from water environment (I). National Institute of Environmental Research 2016:17. Report No. NIER-RP2016-190
  16. Schmidt M, Themann A, Drexler C, Bayer M, Lanzer G, and Menichetti E et al. Blood donor screening for parvovirus B19 in Germany and Austria. Transfusion 2007;47:1775-1782.
    Pubmed CrossRef
  17. Schwarz TF, Roggendorf M, and Deinhardt F. Human parvovirus B19: ELISA and Immunobolt assays. J Virol Methods 1988;20:155-168.
    Pubmed CrossRef
  18. Sevall JS, ritenhous J, and Peter JB. Laboratory diagnosis of parvovirus B19 infection. J Clin Lab Anal 1992;6:171-175.
    Pubmed CrossRef
  19. Sewell DL. Laboratory-associated infections and biosafety. Clin Microbiol Rev 1995;8:389-405.
    Pubmed KoreaMed CrossRef
  20. Smith CJ, and Osborn AM. Advantages and limitations of quantative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 2009;67:6-20.
    Pubmed CrossRef
  21. Nagai K, Horita N, Yamamoto M, Tsukahara T, Nagakura H, and Tashiro K et al. Diagnostic test accuracy of loop-mediated isothermal amplification assay for mycobacterium tuberculosis: systematic review and meta-analysis. Sci Rep 2016;6:39090.
    Pubmed KoreaMed CrossRef
  22. White DG, Woolf AD, Mortimer PP, Cohen BJ, Blake DR, and Bacon PA. Human parvovirus arthropathy. Lancet 1985;1:419-421.
    Pubmed CrossRef
  23. Yamakawa Y, Oka H, Hori S, Arai T, and Izumi R. Detection of human parvovirus B19 DNA by nested polymerase chain reaction. Obstet Gynecol 1995;86:126-129.
    Pubmed CrossRef