© 2011 International Mycological Association
You are free to share - to copy, distribute and transmit the work, under the following conditions:
Attribution: <RXPXVWDWWULEXWHWKHZRUNLQWKHPDQQHUVSHFL¿HGE\WKHDXWKRURUOLFHQVRUEXWQRWLQDQ\ZD\WKDWVXJJHVWVWKDWWKH\HQGRUVH\RXRU\RXUXVHRIWKHZRUN
Non-commercial: <RXPD\QRWXVHWKLVZRUNIRUFRPPHUFLDOSXUSRVHV
No derivative works: <RXPD\QRWDOWHUWUDQVIRUPRUEXLOGXSRQWKLVZRUN
For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights.
INTRODUCTION
)XQJLUHSUHVHQWWKHJUHDWHVWHXNDU\RWLFGLYHUVLW\RQHDUWKDQG WKH\DUHDPRQJWKHSULPDU\GHFRPSRVHUVLQHFRV\VWHPV,WLV FRQVHUYDWLYHO\HVWLPDWHGWKDWPLOOLRQVSHFLHVRIIXQJLH[LVW +DZNVZRUWK0DQ\IXQJDOVSHFLHVDUHLPSRUWDQWSODQW DQG KXPDQ SDWKRJHQV $JULRV 5DSLG DQG DFFXUDWH
detection of fungal pathogens to species or strain level is often essential for disease surveillance and implementing a
GLVHDVH PDQDJHPHQW VWUDWHJ\ 'HYHORSLQJ GLUHFW GHWHFWLRQ
assays is challenging because fungal pathogens can exist as multiple species complexes or at very low concentration
LQ FOLQLFDO DQG QDWXUDO HQYLURQPHQWV 'LIIHUHQW PROHFXODU JHQRW\SHVYDULHWLHV FDQ DOVR H[LVW ZLWKLQ VSHFLHV DQG PD\ KDYH GLIIHUHQW SDWKRJHQLF SUR¿OHV DQG YLUXOHQFH OHYHOV WR WKH KRVW ,Q DGGLWLRQ XQFXOWXUDEOH DQG QRQVSRUXODWLQJ
fungi remain a major challenge when studying biotrophic,
HQGRSK\WLF DQG P\FRUUKL]DO JURXSV 7KHUHIRUH QRYHO
techniques are required when attempting to detect fungi in
WKHHQYLURQPHQW
Increasingly molecular techniques are employed in
VWXGLHVUHTXLULQJWKHGHWHFWLRQRIIXQJLLQWKHHQYLURQPHQW ,Q WKLV SDSHU EDVHG RQ SUHVHQWDWLRQV DW D 6SHFLDO ,QWHUHVW*URXSPHHWLQJFRQYHQHGGXULQJWKH,QWHUQDWLRQDO 0\FRORJLFDO &RQJUHVV ,0& LQ (GLQEXUJK 8. $XJXVW VRPHRIWKHODWHVWGLDJQRVWLFWHFKQLTXHVHPSOR\HG LQ WKH GHWHFWLRQ RI IXQJL LQFOXGLQJ ÀXRUHVFHQFHin situ K\EULGLVDWLRQ ),6+ '1$ DUUD\ WHFKQRORJ\ 0XOWLSOH[ WDQGHP 3&5 DQG 3DGORFN SUREH WHFKQRORJ\ ZLWK UROOLQJ FLUFOH DPSOL¿FDWLRQ DQG ORRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQ /$03 ZHUH GLVFXVVHG 7KH LPSRUWDQFH RI '1$ H[WUDFWLRQ DQG VDPSOLQJ PHWKRGRORJLHV ZHUH DOVR EULHÀ\GLVFXVVHG
7HFKQLVFKH8QLYHUVLWlW%HUOLQ(QYLURQPHQWDO0LFURELRORJ\6HNU)5)UDQNOLQVWUDVVH%HUOLQ*HUPDQ\ &%6.1$:)XQJDO%LRGLYHUVLW\&HQWUH8SSVDODODDQ&78WUHFKW7KH1HWKHUODQGV
'HSDUWPHQWRI3DUDVLWRORJ\DQG0\FRORJ\6FKRRORI0HGLFLQH0DVKKDG8QLYHUVLW\RI0HGLFDO6FLHQFHV0DVKKDG,UDQ
&XUUHQWPDLOLQJDGGUHVV'HSDUWPHQWRI/DERUDWRU\0HGLFLQH&HQWHU'ULYH1DWLRQDO,QVWLWXWHVRI+HDOWK%HWKHVGD0'86$ &XUUHQWPDLOLQJDGGUHVV)HGHUDO(QYLURQPHQW$JHQF\*HUPDQ\&RUUHQVSODW]%HUOLQ*HUPDQ\
Abstract:0DQ\VSHFLHVRIIXQJLFDQFDXVHGLVHDVHLQSODQWVDQLPDOVDQGKXPDQV$FFXUDWHDQGUREXVW
GHWHFWLRQ DQG TXDQWL¿FDWLRQ RI IXQJL LV HVVHQWLDO IRU GLDJQRVLV PRGHOLQJ DQG VXUYHLOODQFH $OVR GLUHFW
detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great
PDQ\IXQJLDUHGLI¿FXOWRULPSRVVLEOHWRFXOWLYDWH,QWKHODVWGHFDGHHIIHFWLYHDPSOL¿FDWLRQSODWIRUPVSUREH GHYHORSPHQWDQGYDULRXVTXDQWLWDWLYH3&5WHFKQRORJLHVKDYHUHYROXWLRQL]HGUHVHDUFKRQIXQJDOGHWHFWLRQ DQGLGHQWL¿FDWLRQ([DPSOHVRIWKHODWHVWWHFKQRORJ\LQIXQJDOGHWHFWLRQDQGGLIIHUHQWLDWLRQDUHGLVFXVVHG KHUH
Article info:6XEPLWWHG2FWREHU$FFHSWHG1RYHPEHU3XEOLVKHG1RYHPEHU
Key words: ),6+ /$03
macroarray medical mycology molecular diagnostics molecular ecology padlock probe pathogenic fungi plant pathology
AR
TICLE
GENERAL CONSIDERATIONS
6XLWDEOH PHWKRGRORJLHV WR LVRODWH QXFOHLF DFLGV IURP WKH
enormous range of habitats that fungi can occupy are crucial
WRDOOPROHFXODUGHWHFWLRQPHWKRGV&ULWLFDOWRWKHVXFFHVVIXO
isolation of nucleic acids are the methods used for extraction and sample preparation, as well as the sampling strategy
HPSOR\HG
DNA extraction methods
$Q HQRUPRXV YDULHW\ RI QXFOHLF DFLG H[WUDFWLRQ PHWKRGV LV DYDLODEOH )RU PDQ\ DSSOLFDWLRQV FRPPHUFLDO NLWV DUH DYDLODEOHEXWWKHVHDUHQRWDOZD\VVXLWDEOH)RUH[DPSOHLQ
the case of soil, none of the kits presently available are able
WR H[WUDFW HI¿FLHQWO\ XVLQJ VDPSOH VL]HV W\SLFDOO\ UHTXLUHG VHH EHORZ 7KHUHIRUH ZLWK XQXVXDO DQG GLI¿FXOW VDPSOH W\SHV FXVWRPL]HG PHWKRGV DUH W\SLFDOO\ HPSOR\HG .H\ WR WKLVLVWKHFHOOGLVUXSWLRQKRPRJHQLVDWLRQVWHSLQWKHSUHVHQFH RI DQ DSSURSULDWH EXIIHU W\SLFDOO\ D &7$% RU JXDQLGLQLXP WKLRF\DQDWHEDVHGEXIIHU$ZLGHYDULHW\RIPHWKRGVFDQEH
used, including specialist equipments such as a planetary
EDOO PLOO %ULHUOH\et al. DSSURSULDWH EHDG EHDWHUV SUHVVXUHF\FOLQJWHFKQRORJ\2NXEDUDet al.DQGQRYHO
approaches using equipment such as a paint shaker for soil
VDPSOHV5HHOHGHUet al.RUHYHQDFRQYHQWLRQDOIRRG EOHQGHUIRUSODQWDQGIRRGVDPSOHV$OOFHOOGLVUXSWLRQVWHSVDUH
potentially damaging to the nucleic acid quantity and quality, either through direct shearing of the nucleic acid, or through
WKHFRH[WUDFWLRQRIKXPLFDFLGVDQLQKLELWRURI3&5SUHVHQW LQSODQWDQGVRLOVDPSOHV7KHUHIRUHPHWKRGVZKLFKSURYLGHDQ
adequate level of cell disruption to enable satisfactory nucleic acid extraction, but without too much nucleic acid damage or
UHOHDVHRISRWHQWLDO3&5LQKLELWRUVDUHUHTXLUHG
)ROORZLQJFHOOGLVUXSWLRQDQGVXEVHTXHQWH[WUDFWLRQVWHSV WKH QXFOHLF DFLG FDQ EH SXUL¿HG XVLQJ VWDQGDUG PHWKRGV LQFOXGLQJ SHOOHWLQJ VLOLFD PHPEUDQH VSLQ ¿OWHU DQG VLOLFD PDJQHWLF SDUWLFOH VHSDUDWLRQ :KHQ VHOHFWLQJ D SXUL¿FDWLRQ
step, consideration needs to be given to the nucleic acid
TXDOLW\TXDQWLW\UHTXLUHGFRVWVSHHGRIH[WUDFWLRQDQGDELOLW\ WRDXWRPDWHDQ\SURFHVV7KHPHWKRGRIDQDO\VLQJWKHQXFOHLF DFLG ZLOO GHWHUPLQH ZKDW SXUL¿FDWLRQ PHWKRG LV XVHG )RU H[DPSOH'1$SUR¿OLQJDQGPHWDJHQRPLFVWXGLHVXVLQJQH[W
generation sequencing approaches will require pure, high
TXDOLW\ QXFOHLF DFLG H[WUDFWV &RQYHUVHO\ FUXGHU OHVV SXUH H[WUDFWVPD\EHXVHGZKHUHUHDOWLPH3&5LVXVHGIRUURXWLQH
diagnostic purposes where the emphasis is on nucleic acid quantity, speed and cost of extraction because the downstream
DSSOLFDWLRQLVPRUHWROHUDQWRILPSXULWLHVLQWKH'1$H[WUDFW
Sample preparation
Innovative sample preparation can enhance chances of
GHWHFWLRQ )RU H[DPSOH RUJDQLF PDWWHU FDQ EH UHPRYHG
from soil and processed separately for fungal targets which
VXUYLYH VROHO\ LQ WKDW FRPSRQHQW RI WKH VRLO 6FOHURWLXP
forming pathogens may be separated from the rest of the soil
E\VLHYLQJRUÀRDWLQJSULRUWRQXFOHLFDFLGH[WUDFWLRQ7KHVH
approaches effectively concentrate the fungal target, thereby
LQFUHDVLQJ WKH FKDQFH RI VXFFHVVIXO GHWHFWLRQ :KHUH
appropriate, a baiting approach could be used in conjunction
ZLWK QXFOHLF DFLG H[WUDFWLRQ DQG 3&5 7KLV KDV WKH DGGHG EHQH¿WRIFRQ¿UPLQJWKHWDUJHWLVYLDEOH
Sampling strategy
In addition, an appropriate and statistically robust sampling
VWUDWHJ\ VKRXOG EH XWLOLVHG 7KLV ZLOO YDU\ EHWZHHQ IXQJDO WDUJHWV GHSHQGLQJ XSRQ WKH ELRORJ\ DQG SRVVLEO\ HSLGHPLRORJ\RIWKHRUJDQLVPWREHGHWHFWHG)RUH[DPSOHD µ:¶VDPSOLQJVWUDWHJ\DFURVVDQDUHDPD\QRWEHDSSURSULDWH
for a target with a highly clustered distribution, and a grid
VDPSOLQJ DSSURDFK VKRXOG EH XVHG LQVWHDG 7KH LQGLYLGXDO VDPSOH VL]H SURFHVVHG IRU '1$ H[WUDFWLRQ VKRXOG DOVR EH ODUJHHQRXJKWREHELRORJLFDOO\UHOHYDQW2SKHO.HOOHUet al. $ZLGHYDULHW\RIPHWKRGVH[LVWDQGQRRQHDSSURDFK LVOLNHO\WREHVXLWDEOHIRUDOOWDUJHWV.QRZOHGJHRIWKHELRORJ\
of the target species is essential for designing and determining the optimum sampling and extraction methodology in any
SDUWLFXODUFDVH
SEQUENCING INDEPENDENT METHODS
'1$VHTXHQFLQJRIWKHLQWHUQDOWUDQVFULEHGVSDFHU,76DQG ODUJHVXEXQLW/68UHJLRQVRIU51$IROORZHGE\FRPSDUDWLYH VHTXHQFHDQDO\VLVKDVEHHQWKHµJROGVWDQGDUG¶IRUPROHFXODU LGHQWL¿FDWLRQRIPRVWIXQJLSDUWLFXODUO\RIFXOWXUDEOHIXQJL7KLV
strategy is fast and accurate, but is dependent on sequence
TXDOLW\ LQ H[LVWLQJ UHIHUHQFH GDWDEDVHV ,Q PDQ\ UHFHQWO\
evolved fungal groups, however, the ribosomal genes are
LQVXI¿FLHQWO\ YDULDEOH DQG VHTXHQFLQJ RI DGGLWLRQDO JHQHV LV QHFHVVDU\ &RVWV FRXOG DOVR EH D FKDOOHQJH IRU URXWLQH
diagnostic laboratories, and therefore various sequencing
LQGHSHQGHQWPHWKRGVWKDWDUHDYDLODEOHFDQEHXVHG
Fluorescent
in situ
hybridisation
6LQFHIXQJLDUHXELTXLWRXVLWLVLPSRUWDQWWRXQGHUVWDQGWKHLU
biodiversity and abundance, as well as their ecological roles in different habitats, such as soil, decaying leaves and wood,
DQGDOVRLQGRRUHQYLURQPHQWVRUKXPDQV,QGHHGLWLVFULWLFDO WR LGHQWLI\ WKH PHWDEROLFDOO\ DFWLYH VSHFLHV µUHDO SOD\HUV¶
in communities or ecosystems in order to understand
WKH HFRV\VWHP SURFHVVHV WKDW LQYROYH IXQJL 3&5 EDVHG PHWKRGVVXFKDV¿QJHUSULQWLQJRUPROHFXODUFORQLQJGRQRW GLVFULPLQDWHEHWZHHQµUHDOSOD\HUV¶DQGIXQJLWKDWDUHGRUPDQW $OVR'1$DQG51$H[WUDFWLRQSURWRFROVPD\EHELDVHGGXH WRULJLGFHOOZDOOVRIIXQJDOK\SKDH,PPXQRVWDLQLQJSURYLGHV
a means to identify species in situ but is extremely laborious in the experimental preparation. $OOWKHVHNLQGVRIWHFKQLFDO GLI¿FXOWLHV FDQ KRZHYHU EH RYHUFRPH E\ ÀXRUHVFHQFHin situK\EULGLVDWLRQ),6+
),6+ LV D SRZHUIXO PHWKRG IRU WKHin situ detection of
RI SDUWLFXODU '1$ RU 51$ VHTXHQFHV LQ WKH F\WRSODVP RUJDQHOOHVRUQXFOHLRIELRORJLFDOPDWHULDOV$VDUHVXOWWKH
technique can detect metabolically active fungi directly in the
HQYLURQPHQW ZLWKRXW FXOWLYDWLRQ ZKHQ 51$ LV SUHVHQW 7KH
spatial distribution of growing mycelia on or within colonized
VXEVWUDWDFDQDOVREHLQYHVWLJDWHG/Let al%DVFKLHQet al0F$UWKXUet al5RELQet al.
7KH PDMRU VWHS RI ),6+ LQYROYHV WKH SUHSDUDWLRQ RI
biological materials or environmental samples, and the
ODEHOOLQJ LQFRUSRUDWLRQ RI D ÀXRUHVFHQW ODEHOPDUNHU RI D QXFOHLFDFLGVHTXHQFHWRIRUPDSUREH7KHQXQGHUFRQWUROOHG H[SHULPHQWDOFRQGLWLRQVWKHSUREHLVK\EULGL]HGWRWKH'1$ RU 51$ LQ ELRORJLFDO PDWHULDOV WR IRUP D GRXEOHVWUDQGHG PROHFXOH )LQDOO\ WKH VLWHV RI K\EULGL]DWLRQ DUH GHWHFWHG DQG YLVXDOL]HG ),6+ LV FRPPRQO\ XVHG LQ WKH µWRSERWWRP
approach’ research followed with feedback to the top level
$PDQQet al 7KH PROHFXODU ELRORJ\ DQG JHQHWLFV
of targeted organism is well-understood, and the probe is designed based on the nucleotide sequence which has been
ZHOOFKDUDFWHUL]HG
),6+ SUREHV RIWHQ WDUJHW VHTXHQFHV RI ULERVRPDO 51$
or mitochondrial genes because they are abundant in
VHTXHQFHGDWDEDVHVDQGLQPXOWLSOHFRSLHVLQHDFKFHOO7KH
probes can be designed by computer-assisted search from
WDUJHW RUJDQLVPV IRU ³VLJQDWXUHV´ 7KHVH VLJQDWXUH UHJLRQV FDQ EH VSHFL¿F DW GLIIHUHQW SK\ORJHQHWLF OHYHOV GHSHQGLQJ RQ WKH YDULDELOLW\ RI WKH WDUJHW PROHFXOH VHTXHQFHV 7KH SUREH FRPSULVHV D VKRUW VHTXHQFH UDQJLQJ IURP WR QXFOHRWLGHV WKDW LV VSHFL¿F IRU RQH RU VHYHUDO WD[D DW VSHFLHV JHQXV RU KLJKHU WD[RQRPLF UDQNV 7KH SUREH RU ROLJRQXFOHRWLGH LV WKHQ ODEHOOHG ZLWK D ÀXRURFKURPH IRU H[DPSOHDFDUERLQGRF\DQLQHG\H&<
$SDUWLFXODUDWWUDFWLRQLVWKDW),6+SUREHVFDQEHDSSOLHG WRIRUPDOGHK\GHRUHWKDQRO¿[HGHQYLURQPHQWDOVDPSOHVRU WRFXOWXUHV$WWKHULERVRPHVWKHSUREHZLOOVSHFL¿FDOO\DQQHDO WRLWVFRPSOHPHQWDU\VHTXHQFHUHVXOWLQJLQDKHWHURGXSOH[ $IWHULQFXEDWLRQDZDVKLQJVWHSLVFUXFLDOWRGLVFULPLQDWHWKH WDUJHW IURP QRQWDUJHW RUJDQLVPV 3UREHV WKDW GR QRW ELQG WR DQ U51$ VHTXHQFH ZLOO EH ZDVKHG DZD\ UHVXOWLQJ LQ D ÀXRUHVFHQWVLJQDORIH[FOXVLYHO\WDUJHWRUJDQLVPV7KHSUREH
conferred signal is correlated with the ribosome content, and
WKHUHIRUHLQFUHDVHVLQFHOOVZLWKKLJKHUPHWDEROLFDFWLYLW\ 7KH ¿UVW ),6+ SUREH WDUJHWLQJ D OLYLQJ IXQJXV ZDV GHVLJQHG E\ /Let al. IRUAureobasidium pullulans RQ WKH SK\OORSODQH RI DSSOH VHHGOLQJV WKLV ZDV WKH ¿UVW WLPH WKDW D OLYLQJ IXQJXV KDG EHHQ YLVXDOL]HG E\ ),6+ ([DPSOHVRIVLWXDWLRQVLQZKLFKWKHPHWKRGKDVEHHQXVHG DUH YHU\ YDULHG )XQJL EHORQJLQJ WREurotiomycetes were demonstrated to be more abundant than Dothideomycetes in
DQH[WUHPHO\DFLGLFPLQHGUDLQDJHXVLQJ6U51$WDUJHWHG SUREHV%DNHUet al%DVFKLHQet al.GHVLJQHG QLQH WD[RQVSHFL¿F SUREHV WDUJHWLQJ WKH 6 RU 6 U51$ IRUWKHGHWHFWLRQRIDTXDWLFK\SKRP\FHWHVLQOHDIOLWWHU)LJ $ % 'RPDLQ VSHFL¿F SUREHV ZHUH DOVR XVHG WR GHWHFW DFWLYHIXQJLLQPLFH6FXSKDPet alDQGLQZDVWHZDWHU VHZDJH JUDQXOH ELR¿OPV :HEHUet al 0RVW SUREHV KDYHEHHQGHVLJQHGWRWDUJHWWKH6RU6U51$JHQHDQG WKHLUVSHFL¿FLW\QHHGVWREHNHSWXQGHUUHYLHZDVVHTXHQFH GDWDEDVHVH[SDQG
6HYHUDOIDFWRUVFDQLQÀXHQFHWKHHI¿FLHQF\RI),6+IRU H[DPSOHWKHVWHULFDODQGHOHFWURVWDWLFDOSURSHUWLHVRIU51$
the features of the probe, and hybridisation conditions such
DV WKH ¿[DWLRQ PHWKRG EXIIHUV WKH VWULQJHQF\ RI SUREH ELQGLQJDQGLQFXEDWLRQWLPH,QDFLRet al.LQYHVWLJDWHG
AR
TICLE
WKH GLIIHUHQW DFFHVVLELOLW\ RI WKH U51$ PROHFXOH IRU ),6+ SUREHV7KH\GHVLJQHGGLIIHUHQWSUREHVWKDWWDUJHWHGWKH 6ODUJHVXEXQLWRIU51$IRU\HDVWVDQGLQYHVWLJDWHGWKH FRQIHUUHG VLJQDO LQWHQVLW\ JHQHUDWHG IURP WKH SUREHV7KH\ FRQFOXGHG WKDW WKH SUHUHTXLVLWH IRU VXFFHVVIXO ),6+ LV WKH VSHFL¿FLW\RISUREHV,QiFLRet al
/LPLWDWLRQV RI WKH ),6+ PHWKRG FDQ LQFOXGH IXQJDO DQG VXEVWUDWHLQKHUHQWDXWRÀXRUHVFHQFHLQVXI¿FLHQWSHUPHDELOLW\ RIFHOOZDOOVQRQVSHFL¿FELQGLQJRISUREHVDQGORZULERVRPH FRQWHQWV $XWRÀXRUHVFHQFH RI IXQJL FDQ DOVR OHDG WR IDOVH SRVLWLYHÀXRUHVFHQFHVLJQDOV$ÀXRUHVFHQFHVFDQFRQGXFWHG
by confocal laser scanning microscopy revealed that many
IUHVKZDWHUIXQJLHPLWDJUHHQDXWRÀXRUHVFHQFH%DVFKLHQet al DQG FRQVHTXHQWO\ D JUHHQODEHOOHG ),6+ SUREH FRXOG QRW EH XVHG ,W LV WKHUHIRUH LPSRUWDQW WR FKHFN IRU DXWRÀXRUHVFHQFH RI ERWK WDUJHW DQG QRQWDUJHW RUJDQLVPV EHIRUH VHOHFWLQJ WKH ÀXRURFKURPH IRU SUREH ODEHOOLQJ (QYLURQPHQWDOVDPSOHVDQGVXEVWUDWHVVXFKDVOHDYHVZRRG DQG DQLPDO WLVVXHV DOVR HPLW VWURQJ DXWRÀXRUHVFHQFH LQ
several wavelengths at the same time, interfering with probe
VLJQDOV+RZHYHUWKHSLQKROHWHFKQLTXHRIDODVHUVFDQQLQJ
microscope is helpful in reducing background emissions
FRPSDUHG WR FRQYHQWLRQDO HSLÀXRUHVFHQFH %DVFKLHQet al
/RZ RU QR SHUPHDELOLW\ RI IXQJDO FHOO ZDOOV FDQ OHDG WR ZHDNRUQRVLJQDOVEHFDXVHRIWKHIDLOXUHRI),6+SUREHVWR SHQHWUDWHULJLGFHOOZDOOV%UXOet al2QHSRVVLEOHZD\
to overcome this problem is to use cell wall lysing enzymes,
VXFKDVFKLWLQDVHVDQGJOXFDQDVHV$PRUHHOHJDQWPHWKRG LVWKHXVHRISHSWLGHQXFOHLFDFLG31$SUREHV31$SUREHV
are mimics in which the negatively charged sugar-backbone
RI'1$LVUHSODFHGZLWKDQRQFKDUJHGSRO\DPLGHEDFNERQH 31$ SUREHV VKDUH WKH VDPH QXFOHRWLGH EDVHV DV WKH FRQYHQWLRQDO'1$SUREHV+RZHYHU31$SUREHVSHQHWUDWH
cell walls more effectively due to their neutrality and they do not have to overcome the destabilizing electrostatic repulsion
GXULQJK\EULGLVDWLRQ:LOVRQet al6KHSDUGet al /RZULERVRPHFRQWHQWVFDQDULVHIURPHLWKHUDVFDUFLW\RI
potential substrate in oligotrophic habitats, or the presence of
FRPSRQHQWVLQKLELWLQJIXQJDOJURZWK+RZHYHUWKH&DWDO\VHG 5HSRUWHU 'HSRVLWLRQ ),6+ &$5'),6+ LV D YDULDQW RI WKH ),6+PHWKRGGHVLJQHGWRHQKDQFHSUREHFRQIHUUHGVLJQDOV 7KHPDMRUGLIIHUHQFHLVWKDWWKHSUREHVDUHODEHOOHGZLWKKRUVH UDGLVKR[LGDVH LQVWHDG RI D ÀXRURFKURPH )OXRUHVFHQFH ODEHOOHGW\UDPLGHLVWKHQDGGHGWRWKHFHOOVDIWHUK\EULGLVDWLRQ 7KH KRUVHUDGLVKR[LGDVH FDWDO\VHV WKH GHSRVLWLRQ RI WKH UHSRUWHUDQGWKLVUHDFWLRQOHDGVWRWRIROGVWURQJHU),6+ VLJQDOVWKDQFRQYHQWLRQDO),6+&RQVHTXHQWO\WKHQXPEHURI DFWLYHU51$PROHFXOHVQHFHVVDU\WRGHWHFWDSUREHFRQIHUUHG VLJQDO GHFUHDVHV E\ XVLQJ &$5' ),6+ 3HUQWKDOHUet al
DNA array hybridization
'1$ DUUD\ K\EULGL]DWLRQ, also NQRZQ DV 5HYHUVH 'RW %ORW +\EULGL]DWLRQ 5'%+ RU PDFURDUUD\ LV D WHFKQLTXHbased on K\EULGL]DWLRQ RI DPSOL¿HG DQG ODEHOOHG JHQRPH UHJLRQV of interest to immobilized oligonucleotides spotted on a
VROLGVXSSRUWSODWIRUPItwas originally developed to detect mutations of human genes, and is still an important diagnostic
WRRO LQ FOLQLFDO UHVHDUFK &KHKDE :DOO <DQJet al =KDQJet al. ,W LV QRZ FRQVLGHUHG D SRZHUIXO DQG SUDFWLFDO WHFKQLTXH IRU WKH GHWHFWLRQ DQG LGHQWL¿FDWLRQ
of fungi and other microbes, such as bacteria, from complex environmental samples without the need for isolation in
FXOWXUH&KHQet al(KUPDQQet al/pYHVTXHet al. 7DPERQJet al. 8HKDUDet al:Xet al =KDQJet al. )RUWKLVW\SHRIDSSOLFDWLRQ
oOLJRQXFOHRWLGHVRUPLFURFRGHV6XPPHUEHOOet alDUH designed from a taxonomically complete dataset of suitable JHQRPHUHJLRQV&KHQet al7DPERQJet al 7KHROLJRQXFOHRWLGHVFDQEHVHOHFWHGPDQXDOO\E\DQDO\VLQJ multi-sequence alignments, or through computer programs, VXFK DV 6LJ2OL=DKDULHYet al DQG $UUD\ 'HVLJQHU 3UHPLHU %LRVRIW ,QWHUQDWLRQDO3DOR $OWR &$ 6\QWKHVL]HG ROLJRQXFOHRWLGHVZLWK¶DPLQHPRGL¿FDWLRQVDUHWKHQVSRWWHG onto a supporting platform, such as a nylon membrane or JODVV VOLGH HLWKHU PDQXDOO\ RU URERWLFDOO\ 5RERWLF VSRWWLQJ can VLJQL¿FDQWO\LQFUHDVHWKHPD[LPXPGHQVLW\RIWKHDUUD\
which can favour the detection of broader taxonomic groups
&KHQet al $PSOLFRQV RI WKH WDUJHW JHQH UHJLRQV DUH DPSOL¿HG E\ 3&5 ODEHOOHG ZLWK GLJR[\JHQLQ ',* DQG VXEMHFWHG WR WKH '1$ K\EULGL]DWLRQ SURFHGXUH SUHYLRXVO\ GHVFULEHG)HVVHKDLHet al.$SRVLWLYHUHDFWLRQEHWZHHQ DQ DPSOLFRQ DQG D SHUIHFWO\ PDWFKHG 30 ROLJRQXFOHRWLGH
generates a chemiluminescent signal which can be detected
E\ ;UD\ ¿OP RU D GLJLWDO FDPHUD LQ GDUN URRPV &DSWXUHG
images are then analysed on a computer program such as
*HQH3L[3UR0ROHFXODU'HYLFHV6XQQ\YDOH&$
7KHGHVLJQRIVSHFLHVRUJURXSVSHFL¿FROLJRQXFOHRWLGHV LVDFUXFLDOVWHSLQWKLVSURFHVVVLQFHLWGH¿QHVWKHVSHFL¿FLW\ DQGVHQVLWLYLW\RIWKHDVVD\/LHYHQVet al3R]KLWNRY et al 8UDNDZDet al It is generally agreed that the length of the oligonucleotide, the number, type and
SRVLWLRQRI613VFRQWDLQHGLQDQROLJRQXFOHRWLGHGHWHUPLQH LWVGLVFULPLQDWRU\SRWHQWLDOIRU'1$K\EULGL]DWLRQ$GHVLUDEOH ROLJRQXFOHRWLGH VKRXOG KDYH VXLWDEOH WKHUPRG\QDPLF FKDUDFWHUV VXFK DV PHOWLQJ WHPSHUDWXUH FRQWDLQ DV
many polymorphic sites as possible located close to the
FHQWUH RU RQ WKH ¶KDOI RI WKH VHTXHQFH DQG KDYH WKH OHDVWSUREDELOLW\RIKDLUSLQRUGLPHUIRUPDWLRQ.DZDVDNLet al /LHYHQVet al2OLJRQXFOHRWLGHOHQJWKVUDQJLQJ IURPWRPHUKDYHGLVSOD\HGWKHEHVWEDODQFHEHWZHHQ VSHFL¿FLW\DQGVHQVLWLYLW\&KHQet al/RQJHUOHQJWKV !PHURIROLJRQXFOHRWLGHVUHGXFHWKHUDWLRRIPLVPDWFKHG
to matched base pairs, yet increase the number of bases
DYDLODEOH IRU K\EULGL]DWLRQ SURYLGLQJ ORZHU VSHFL¿FLW\ EXW KLJKHUVHQVLWLYLW\WRDQDUUD\'RUULVet al/LHYHQVet al 2OLJRQXFOHRWLGHV XS WR PHU KRZHYHU KDYH EHHQ
used in macroarrays for the detection of plant viruses using gene sequences that are widely different between the closely
UHODWHG SDWKRJHQV $JLQGRWDQ 3HUU\ ,W KDV
in affecting the discriminatory power of an oligonucleotide
8UDNDZDet al /LHYHQVet al 3R]KLWNRYet al 'HVSLWHQXPHURXVDWWHPSWVWRSUHGLFWWKHEHKDYLRXU RIROLJRQXFOHRWLGHVLQ'1$K\EULGL]DWLRQWKHWKHUPRG\QDPLF
interaction between the probes and oligonucleotides remains
SRRUO\XQGHUVWRRG3R]KLWNRYet al
0RVW '1$ PDFURDUUD\V WKDW KDYH EHHQ GHYHORSHG DUH EDVHG RQ D VLQJOH UHJLRQ IRU WKH GHWHFWLRQ RI D VSHFL¿F WD[RQRPLFJURXS$PRQJWKHVHUHJLRQV6ULERVRPDO'1$ KDV EHHQ XVHG IRU WKH GHWHFWLRQ RI EDFWHULD )HVVHKDLHet al;LRQJet al.9DULRXVJHQRPHUHJLRQVVXFK DVULERVRPDO'1$VSDFHUV,76PLWRFKRQGULDOJHQHVHJ F\WRFKURPH R[\GDVH F VXEXQLW cox DQG VRPH SURWHLQ FRGLQJUHJLRQVȕWXEXOLQ()ĮHWFZHUHFKRVHQWRWDUJHW IXQJLDQGIXQJXVOLNHRUJDQLVPV&KHQet al.*LOEHUWet
al+DUSHUet al1LQJet al6HLIHUW
/pYHVTXH 2OLJRQXFOHRWLGHV ZLWK KLJKHU VSHFL¿FLW\
are often designed from polymorphic sites located at indels
SUHVHQWLQPXOWLVHTXHQFHDOLJQPHQWV5RELGHDXet al 7DPERQJet al 2OLJRQXFOHRWLGHV H[WUDFWHG IURP D
locus that can be well aligned with low sequence divergence tends to cross-react with non-target amplicons and display strong false positive signals even when tested with pure cultures because of the low frequency of polymorphisms
LQ WKH VSHFL¿F ROLJRQXFOHRWLGHV7KLV KDV EHHQ REVHUYHG LQ
arrays using cox LQPenicillium VXEJHQPenicillium &KHQ et al 6HLIHUWet al ,Q D UHFHQW VWXG\ '1$
arrays were constructed from multiple loci of Phytophthora
species, including ITS, coxDQGWKHLQWHUJHQLFUHJLRQcox VSDFHUCSEHWZHHQF\WRFKURPHFR[LGDVHcoxDQG cox : &KHQet al SHUV FRPP ,Q FRPSDULVRQ WR WKH coxUHJLRQWKHOHQJWKYDULDWLRQDQGWKHSUHVHQFHRILQGHOV
in the sequence alignments of ITS and CS provided better
RSSRUWXQLWLHV WR VHOHFW KLJKO\ VSHFL¿F ROLJRQXFOHRWLGHV 7KH
combination of all three arrays increased the discrimination
SRWHQWLDOGHWHFWLRQDFFXUDF\DQGUHGXQGDQF\RIWKHDVVD\ '1$ DUUD\V KDYH EHHQ GHYHORSHG IRU WKH GHWHFWLRQ RI
plant pathogens in a wide range of environmental samples,
VXFKDVJUHHQKRXVHFURSV/H)ORFKet al/LHYHQVet
al.SRWDWRHV)HVVHKDLHet alJLQVHQJ3XQMD et alDQGIUXLWV5RELGHDXet al6KROEHUJet al 0DFURDUUD\VDUHDOVRHIIHFWLYHGLDJQRVWLFWRROV IRUWKHGHWHFWLRQRISK\WRSDWKRJHQLFEDFWHULD)HVVHKDLHet alIXQJLDQGIXQJXVOLNHRUJDQLVPV&KHQet al /pYHVTXHet al 7DPERQJet al QHPDWRGHV 8HKDUDet alDQGYLUXVHV$JLQGRWDQ 3HUU\
'1$ DUUD\ K\EULGL]DWLRQ LV KLJKO\ VHQVLWLYH DV DUH PRVW 3&5EDVHG DSSURDFKHV :LWK WKH XQOLPLWHG FDSDFLW\ IRU
the accommodation of oligonucleotides on one membrane and the reusability of the membranes, it shows superior multiplexing detection capability at a lower cost over other
3&5EDVHG PHWKRGV ,Q D ELRGLYHUVLW\ VWXG\ WKH VSHFLHV SUR¿OH FRXOG EH UHYHDOHG E\ K\EULGL]LQJ WKH ROLJRQXFOHRWLGH DUUD\ ZLWK D PL[HG SRRO RI ',*ODEHOOHG 3&5 SURGXFWV DPSOL¿HGIURPWKHWRWDO'1$RIDTXHU\VDPSOH7KLVDVVD\
would be especially valuable for the simultaneous detection of multiple plant pathogens which cover a broad taxonomic
UDQJH EXW DUH VSHFL¿F WR D FHUWDLQ KRVW $V DQ H[DPSOH 5RELGHDXet alGHYHORSHGD'1$DUUD\IRUFUDQEHUU\ IUXLWURW&)5IXQJDOSDWKRJHQVZLWKRYHU¿HOGVDPSOHV EHLQJWHVWHG7KLVDUUD\KDGWKHDELOLW\WRGHWHFWVSHFLHVIURP JHQHUD RI IXQJL NQRZQ RQ FUDQEHUU\ ZLWK D VLQJOH WHVW )LJ VKRZV WKDW WKLV '1$ PDFURDUUD\ ZDV DEOH WR GHWHFW
species of Phyllosticta, Cladosporium, Epicoccum, Godronia,
Alternaria, Pestalotia, and Pilidium from a single frozen
FUDQEHUU\IUXLWVDPSOH5RELGHDXet al
7KHPDFURDUUD\WHFKQRORJ\LVQRZDYDLODEOHFRPPHUFLDOO\ LQ IRXU (XURSHDQ FRXQWULHV XQGHU WKH QDPH '1$ 0XOWLVFDQ KWWSZZZGQDPXOWLVFDQFRP DQG DW WKH *XHOSK 3HVW 'LDJQRVWLF&OLQLFKWWSZZZJXHOSKODEVHUYLFHVFRPIRU
the test of plant pathogens in greenhouse nutrient solutions
DQGURRWVRIFURSV7KHUHDUHYHU\VLJQL¿FDQWDGYDQFHVLQQH[W
generation sequencing technology that have reduced the need
RI '1$ DUUD\ WHFKQRORJ\ LQ IXQFWLRQDO JHQRPLFV +RZHYHU
there are developments in lab-on-a-chip array technology for
YHU\ TXLFN DQG IRU ODUJHVFDOH WHVWLQJ $ UHFHQW H[DPSOH LV
a low density array for Phytophthora detection, where
amino-Fig. 2.'1$PDFURDUUD\K\EULGL]DWLRQUHVXOWVIURPIUR]HQFUDQEHUU\IUXLWVDPSOHFROOHFWHGIURP0DVVDFKXVHWWV86$DGDSWHGIURP5RELGHDX
et al7KH[UD\¿OPLVRYHUODLGZLWKWKHROLJRQXFOHRWLGHVSRWWLQJSDWWHUQRQ'1$DUUD\PHPEUDQHIRUFUDQEHUU\IUXLWURWIXQJL7KHWRS OHIWSDLURIVSRWVDQGERWWRPULJKWSDLURIVSRWVDUHROLJRQXFOHRWLGHVZKLFKVHUYHGDVSRVLWLYHFRQWUROV7KLVDUUD\LVVKRZLQJSRVLWLYHVLJQDOIRU
AR
TICLE
labelled oligonucleotides are spotted over a gap between two
HOHFWURGHVLQVLGHDPLFURFKLS-XOLFKet al+\EULGL]DWLRQ
is detected by a current passing through a silver deposit over
WKHJDS%HFDXVHRIVXFKGHYHORSPHQWVDUUD\EDVHGDVVD\V DUHOLNHO\WRUHPDLQDSRSXODURSWLRQIRUGLDJQRVWLFV
Multiplex tandem PCR
0XOWLSOH[HGWDQGHP3&5073&5LVDWHFKQRORJ\SODWIRUP GHYHORSHG IRU KLJKO\ PXOWLSOH[HG JHQH H[SUHVVLRQ SUR¿OLQJ DQGWKHUDSLGLGHQWL¿FDWLRQRIFOLQLFDOO\LPSRUWDQWSDWKRJHQV 6WDQOH\et al 7KH SODWIRUP FRQVLVWV RI WZR URXQGV RIDPSOL¿FDWLRQ)LJ,QWKH¿UVWVWHSDPXOWLSOH[3&5LV SHUIRUPHG DW WR F\FOHV WR DOORZ HQULFKPHQW RI WDUJHW '1$ ZLWKRXW FUHDWLQJ FRPSHWLWLRQ EHWZHHQ DPSOLFRQV /DX et al7KLVSURGXFWLVGLOXWHGDQGXVHGDVWHPSODWHIRU WKH VHFRQG DPSOL¿FDWLRQ WKDW FRQVLVWV RI PXOWLSOH LQGLYLGXDO TXDQWLWDWLYH 3&5 UHDFWLRQV ZLWK SULPHUV QHVWHG ZLWKLQ WKRVH XVHG LQ WKH PXOWLSOH[ 3&5 8S WR GLIIHUHQW 3&5 UHDFWLRQVFDQEHPXOWLSOH[HGDQGSHUIRUPHGVLPXOWDQHRXVO\ )OXRUHVFHQFH LV PHDVXUHG E\ 6<%5 JUHHQ WHFKQRORJ\ DW
the end of each extension cycle, and melt-curve analysis
SURYLGHV VSHFLHVVSHFL¿F RU JHQHVSHFL¿F LGHQWL¿FDWLRQ 7KHLQFRUSRUDWLRQRIWZRVHWVRIVSHFLHVVSHFL¿FSULPHUVIRU HDFKWDUJHWHQVXUHVFRUUHFWDPSOL¿FDWLRQDQGGHWHFWLRQWKXV DYRLGLQJWKHH[SHQVHRI'1$SUREHV6<%5JUHHQGHWHFWLRQ
also increases the multiplexing and quantitative capacity
RI UHDOWLPH 3&5 V\VWHPV ZKLFK DUH XVXDOO\ OLPLWHG E\ WKH DYDLODELOLW\RIÀXRUHVFHQWFKDQQHOVDQGWKHQHHGWRRSWLPL]H HDFKLQGLYLGXDOPXOWLSOH[3&5
,Q WKH FOLQLFDO VHWWLQJ LQYDVLYH IXQJDO LQIHFWLRQV ,),V
remain a leading cause of morbidity and mortality in
LPPXQHFRPSURPLVHG KRVWV 073&5 ZDV FRQVLGHUHG D VXLWDEOHDOWHUQDWLYHIRUWKHUDSLGGHWHFWLRQDQGLGHQWL¿FDWLRQ
of fungal pathogens directly from clinical specimens, thus circumventing the need for gold standard culture and
KLVWRORJ\ZKLFKLVVORZLQVHQVLWLYHDQGQRQVSHFL¿F)DVWHU DQG DFFXUDWH PROHFXODU LGHQWL¿FDWLRQ ZRXOG DOVR HQDEOH
better guidance and earlier administration of targeted
DQWLIXQJDO WKHUDSLHV $V VXFK WKH ODERUDWRU\ DW :HVWPHDG KRVSLWDO LQ FRQMXQFWLRQ ZLWK $XV'LDJQRVWLFV $OH[DQGULD 16: $XVWUDOLD GHYHORSHG VHYHUDO 073&5 DVVD\V WR GHWHFWWKHPDMRUFDXVHVRIIXQJDOEORRGVWUHDPLQIHFWLRQV 7KH WDUJHWV LQFOXGHG VSHFLHV RICandida, Cryptococcus neoformans complex, Saccharomyces cerevisiae,
pan-Fusarium, F. solani, and 6FHGRVSRULXP SUROL¿FDQV 3ULPHUV ZHUH GHVLJQHG XVLQJ VHTXHQFH YDULDWLRQV ZLWKLQ WKH ,76 UHJLRQVHORQJDWLRQIDFWRU_()_DQG`WXEXOLQJHQHV
)XQJDO WDUJHWV ZHUH VHOHFWHG DFFRUGLQJ WR WKHLU IUHTXHQF\
of causing infections, their potential resistance to frontline
DQWLIXQJDO DJHQWV HVSHFLDOO\ ÀXFRQD]ROH DQG WKHLU KLJK DWWULEXWDEOHPRUWDOLW\
7KH073&5SODWIRUPZDVLQLWLDOO\HYDOXDWHGRQEORRG FXOWXUHVLQZKLFKD\HDVWRUPRXOGZDVVHHQLQ*UDP¶VVWDLQ SUHSDUDWLRQVDVZHOODVEDFWHULDOEORRGFXOWXUHVDQG VDPSOHVZKLFKGLGQRWÀDJSRVLWLYH/DXet alD7KH VHQVLWLYLW\DQGVSHFL¿FLW\RIWKHDVVD\ZDV7KLVLQFOXGHG WKHFRUUHFWLGHQWL¿FDWLRQRIIXQJLLQ¿YHFDVHVZLWKEDFWHULDO FRLQIHFWLRQ ,Q DGGLWLRQ QR LQWHUIHUHQFH ZDV REVHUYHG LQ VLPXODWHGFDVHVRISRO\IXQJDOLQIHFWLRQ8QIRUWXQDWHO\WKUHH UDUHGLVHDVHFDXVLQJ\HDVWVCandida lambica, C. nivariensis
and Kodamaea ohmeri ZHUH QRW GHWHFWHG E\ 073&5 DV WDUJHWVZHUHQRWDYDLODEOHRQWKHIXQJDOSDQHO1HYHUWKHOHVV
this study demonstrated the diagnostic usefulness of the platform to rapidly identify common fungal pathogens within
IRXU KRXUV RI EORRG FXOWXUH ÀDJJLQJ LQFOXGLQJ DXWRPDWHG QXFOHLFDFLGH[WUDFWLRQZKLFKLVFRQVLGHUDEO\IDVWHUWKDQWKH KUHTXLUHGE\JROGVWDQGDUGPHWKRGV
$QH[SDQGHGYHUVLRQRIWKH073&5IXQJDOSODWIRUPZDV WKHQHYDOXDWHGRQ('7$ZKROHEORRGVHUXPDQG SODVPD VSHFLPHQV REWDLQHG IURP SDWLHQWV ZLWK SURYHQ FDQGLGHPLD/DXet alZLWKWKHDLPRIFLUFXPYHQWLQJ
the technical and sensitivity issues inherent in routine blood
FXOWXUH GLDJQRVLV $OWKRXJK WKH 073&5 DVVD\ ZDV OHVV VHQVLWLYHWKDQEORRGFXOWXUHVHQVLWLYLW\WKHGLDJQRVLV RIFDQGLGHPLDDQGSDWKRJHQLGHQWL¿FDWLRQZDVH[SHGLWHGE\ XS WR IRXU GD\V 7KH UHVXOWV DOVR VXSSRUWHG WKH FRQWHQWLRQ
that serum and plasma samples were better than whole
EORRGVDPSOHVIRUWKHPROHFXODUGHWHFWLRQRIFDQGLGHPLD 8VLQJ D WHFKQLTXH NQRZQ DV FRORQ\ 073&5 /DXet al E WKH IXQJDO DVVD\ ZDV DOVR DEOH WR SURYLGH UDSLG K DQG XQDPELJXRXV LGHQWL¿FDWLRQ RI \HDVWV GLUHFW IURP SULPDU\LVRODWLRQSODWHVZLWKRXWWKHQHHGIRU'1$H[WUDFWLRQRU VHSDUDWLRQIURPPL[HGIXQJDOEDFWHULDOFXOWXUHV$VVXFKFRORQ\ 073&5RIIHUHGDIDVWHUDQGEHWWHUDOWHUQDWLYHWRELRFKHPLFDO DVVD\VZKLFKDUHRIWHQVXEMHFWLYHSURQHWRPLVLGHQWL¿FDWLRQ DQGGHSHQGHQWRQDSXUHFXOWXUHEHLQJREWDLQHG&XUUHQWZRUN LV DLPHG DW LQWHJUDWLQJ LGHQWL¿FDWLRQ SDQHOV ZLWK WDUJHWV WR GHWHFWPROHFXODUPHFKDQLVPVRIDQWLIXQJDOUHVLVWDQFH
2YHUDOOWKHIXQJDO073&5DVVD\FRPSDUHVIDYRXUDEO\
to other commercial platforms and integrates well into the
Fig. 3. 6FKHPDWLF UHSUHVHQWDWLRQ RI WKH PXOWLSOH[ WDQGHP±3&5 SURFHGXUHLOOXVWUDWLQJVSHFL¿FGHWHFWLRQDQGLGHQWL¿FDWLRQIURPDEORRG
URXWLQH ZRUNÀRZ RI GLDJQRVWLF ODERUDWRULHV $XWRPDWHG
operations and use of commercial reagents further enables
VWDQGDUGL]HGSURFHGXUHVWREHHVWDEOLVKHG1HYHUWKHOHVVWKH
major limitation of all molecular diagnostic assays is target availability, which is often dictated by an effort to balance out
FRVWV DQG WXUQDURXQG WLPH ZLWK PD[LPXP WKURXJKSXW7KH
increased sensitivity of molecular assays and its ability to detect viable and non-viable cells should also place heavy emphasis on interpreting results with other microbiological
GDWDDQGFOLQLFDOLQIRUPDWLRQ
ISOTHERMAL SYSTEMS
Isothermal systems do not require a thermal cycler to produce rapid temperature changes, but require only a simple platform
VXFK DV KHDWLQJ EORFNV RU ZDWHU EDWK ,VRWKHUPDO V\VWHPV LQFOXGHUROOLQJFLUFOHDPSOL¿FDWLRQ5&$DQGORRSPHGLDWHG LVRWKHUPDODPSOL¿FDWLRQ/$03
Padlock probe technology and rolling circle
DPSOL¿FDWLRQ
'HWHFWLRQ DQG FKDUDFWHUL]DWLRQ RI VLQJOH QXFOHRWLGH SRO\PRUSKLVPV 613V LV EHFRPLQJ LQFUHDVLQJ SRSXODU IRU SDWKRJHQ LGHQWL¿FDWLRQ EXW ZDV FRQVLGHUHG D PDMRU FKDOOHQJH IRU FRQYHQWLRQDO UHDOWLPH 3&5 XVLQJ UHJXODU ROLJRQXFOHRWLGHV GHWHFWHG E\ ÀXRUHVFHQW G\HV HJ 6<%5 JUHHQRU7DT0DQSUREHV,QRUGHUWRUHFRJQL]H613VDPRQJ GLIIHUHQWJHQRW\SHVSDGORFNSUREHWHFKQLTXHVDUHUHTXLUHG 3DGORFNSUREHV3/3VDUHORQJROLJRQXFOHRWLGHVDERXW ES FDUU\LQJ D QRQWDUJHWFRPSOHPHQWDU\ VHJPHQW ÀDQNHG E\WKHWDUJHWFRPSOHPHQWDU\UHJLRQVDWWKHLU¶DQG¶HQGV ZKLFK UHFRJQL]H DGMDFHQW VHTXHQFHV RQ WKH WDUJHW '1$ 1LOVVRQet al 7KXV RQ K\EULGL]DWLRQ WKH HQGV RI
the probes occupy adjacent positions, and can be joined by
HQ]\PDWLFOLJDWLRQ)LJV±/LJDWLRQRFFXUVDQGWKHSUREHV
are circularized only when both end segments correctly
UHFRJQL]HWKHWDUJHWVHTXHQFHV/DQGHJUHQet al7KH KHOLFDOQDWXUHRIGRXEOHVWUDQGHG'1$GV'1$HQDEOHVWKH
Fig. 4.6FKHPDWLFRYHUYLHZRIWKHSDGORFNSULQFLSOHFRPELQHGZLWK2SHQ$UUD\®7HFKQRORJ\IRUPXOWLSOH[GHWHFWLRQRIWKUHHGLIIHUHQWWDUJHWV
AR
TICLE
probe to topologically bind to the target strand and the probe
FDQ¶WEHGLVSODFHG1LOVVRQet al
3DGORFN SUREHV ZHUH LQLWLDOO\ LQWURGXFHG IRU in situ '1$ ORFDOL]DWLRQ DQG GHWHFWLRQ 1LOVVRQet al 7KH\
were developed originally for discrimination of centromeric
VHTXHQFH YDULDWLRQ LQ KXPDQ FKURPRVRPHV 1LOVVRQet al +RZHYHU WKH PHWKRG KDV QRZ EHHQ DSSOLHG WR GHWHFWLRQ RI JHQHWLFDOO\ PRGL¿HG RUJDQLVPV 3ULQVet al 7KLV FRQFHSW DOVR SURYLGHV H[WHQVLYH PXOWLSOH[
potential for pathogen detection as the interaction between padlock probes does not give rise to circular molecules,
which can be easily removed from the detection system
1LOVVRQet al ) 5HFHQWO\ SDGORFN SUREHEDVHG
applications for multiplex quantitative targets detection and for genotyping fungal and microbial community analysis
XVLQJKLJKWKURXJKSXWUHDOWLPH3&5RQ2SHQ$UUD\V® have
EHHQ GHYHORSHG YDQ 'RRUQet al $GYDQWDJHV RI 3/3EDVHGGLDJQRVWLFDSSOLFDWLRQVGHYHORSHGDUHDÀH[LEOH DQG HDVLO\ DGDSWDEOH GHVLJQ VSHFL¿FLW\ DQG PXOWLSOH[LQJ
universal downstream processing after ligation, and
high-WKURXJKSXW IRUPDW ZLWK UHDOWLPH DQDO\VLV 7VXLet al. )LJ
%ULHÀ\ YDULRXV SDGORFN SUREHV DUH GHVLJQHG WR WDUJHW RUJDQLVPV DQG OLJDWHG WR '1$ H[WUDFWHG IURP HQYLURQPHQWDO VDPSOHV RU FXOWXUHV YDQ 'RRUQet al 7DUJHWV IRU OLJDWLRQSUHVHQWLQFRPSOH['1$VDPSOHVVXFKDVVRLORUUH FLUFXODWHGZDWHUFDQEHJHQHUDWHGE\3&5SUHDPSOL¿FDWLRQ
through Phi29SRO\PHUDVHDQGZKROHJHQRPHDPSOL¿FDWLRQWR HQVXUHHI¿FLHQWGHWHFWLRQ5HDOWLPHTXDQWL¿FDWLRQIRUPXOWLSOH WDUJHWVLVSHUIRUPHGLQ2SHQ$UUD\V®, which can accommodate
XSWR[QO3&5DPSOL¿FDWLRQZLWKSUHORDGHG SUREH VSHFL¿FSULPHUV)LJ0XOWLSOH[SDGORFNOLJDWLRQLVIROORZHG E\VLQJOHSOH[DPSOL¿FDWLRQXVLQJXQLTXHSUREHVSHFL¿FSULPHU SDLUV DQG 6<%5 JUHHQ EDVHG GHWHFWLRQ LQ QDQROLWUH 3&5 DUUD\YDQ'RRUQet al7KHSHUIRUPDQFHRIWKHSDGORFN SUREH GHWHFWLRQ V\VWHP KDV EHHQ GHPRQVWUDWHG XVLQJ
probes targeting several plant pathogens at various taxonomic
OHYHOV 6]HPHVet al YDQ 'RRUQet al $OO SUREHVVSHFL¿FDOO\GHWHFWHGWKHLUFRUUHVSRQGLQJWDUJHWVDQG
provided perfect discrimination against non-target organisms
ZLWK YHU\ VLPLODU WDUJHW VLWHV 3DWKRJHQ TXDQWL¿FDWLRQ ZDV
robust in single target versusPL[HGWDUJHWDVVD\V7KLVQRYHO DVVD\ HQDEOHV YHU\ VSHFL¿F KLJKWKURXJKSXW TXDQWLWDWLYH
detection of multiple pathogens over a wide range of target concentrations, and should be easily adaptable for versatile
GLDJQRVWLFSXUSRVHVYDQ'RRUQet al
$OVRSDGORFNSUREHVFRQWDLQLQJ]LSFRGHVHTXHQFHRUD
biotin-labelled moiety and internal endonuclease cleavage
VLWH LQ FRQMXQFWLRQ ZLWK TXDQWLWDWLYH 3&5 DQG /XPLQH[70
technology or microarray technology, can be used for
PXOWLSOH[SDWKRJHQGHWHFWLRQDQGTXDQWL¿FDWLRQ6]HPHVet al(ULNVVRQet alYDQ'RRUQet al.
$OWHUQDWLYHO\ WKH VLJQDO E\ ZKLFK WKH WDUJHW K\EULGL]HV SHUIHFWO\ WR WKH SDGORFN SUREHV FDQ EH DPSOL¿HG E\ K\SHUEUDQFKHG UROOLQJ FLUFOH DPSOL¿FDWLRQ +5&$ %DQpU et al 5ROOLQJ FLUFOH DPSOL¿FDWLRQ 5&$ LV EDVHG RQ WKHUROOLQJUHSOLFDWLRQRIVKRUWVLQJOHVWUDQGHG'1$VV'1$ FLUFXODUPROHFXOHV/L]DUGLet al, )LUH ;X)5&$
involves a single forward primer complementary to the linker
UHJLRQRIWKHSDGORFNSUREHDQGD'1$SRO\PHUDVHZLWKVWUDQG GLVSODFHPHQW DFWLYLW\ LQ DQ LVRWKHUPDO FRQGLWLRQ 3LFNHULQJ et al $V D UHVXOW WKH SDGORFN SUREH VLJQDO FDQ EH DPSOL¿HG VHYHUDO WKRXVDQGIROG EHFDXVH WKH SRO\PHUDVH
extends the bound primer along the padlock probes for many cycles and displaces upstream sequences, producing a long
VV'1$ PROHFXOH FRPSULVLQJ PXOWLSOH UHSHDWV RI WKH SUREH VHTXHQFH7ZRSULPHUVDUHHPSOR\HGD¿UVWIRUZDUGSULPHU WKDW ELQGV WR WKH SDGORFN SUREH DQG LQLWLDOL]HV 5&$ DQG D
Fig. 5. 6FKHPDWLF UHSUHVHQWDWLRQ RI WKH VWHSV LQ SDGORFN SUREH WHFKQRORJ\ FRXSOHG ZLWK K\SHUEUDQFKHG UROOLQJ FLUFOH DPSOL¿FDWLRQ +5&$IRU613VGHWHFWLRQ1. 7KHK\EULGL]DWLRQRISDGORFNSUREHV FRQWDLQLQJWKHFRPSOHPHQWDU\VHTXHQFHVDWWKH¶DQG¶HQGVWR WKHWDUJHWWHPSODWHV2.'XULQJDSHUIHFWPDWFKWKHSUREHIRUPVD FLUFXODU PROHFXODU ZLWK WKH DLG RI '1$ OLJDVH ZKLOH LQ WKH FDVH RI PLVPDWFKQRFLUFXODUPROHFXOHVIRUPHG3. 1RQK\EULGL]HGWHPSODWH ZLOO EH UHPRYHG GXULQJ WKH H[RQXFOHRO\VLV UHDFWLRQV GLJHVWLRQ E\ H[RQXFOHDVHV , DQG ,,,4. +5&$ LV SHUIRUPHG XVLQJ WZR SUH
VHFRQGSULPHUWKDWWDUJHWVWKHUHSHDWHGVV'1$VHTXHQFHRI WKHSULPDU\5&$SURGXFW¿QDOO\JHQHUDWLQJODUJHQXPEHUVRI FRSLHVRIWKH'1$IUDJPHQWV7KLVLVFDOOHGK\SHUEUDQFKLQJ 5&$+5&$/L]DUGLet al)LJ
3DGORFN SUREH FRXSOHG ZLWK +5&$ RIIHUV D VLJQL¿FDQW DGYDQWDJH IRU WKH GHWHFWLRQ RI 613V 7VXLet al. 7KH
formation of circular probes via ligation occurs when both ends of the padlock probes perfectly hybridize to the target at
MX[WDSRVLWLRQ)LJ7KHVXEVHTXHQW+5&$DPSOL¿FDWLRQRI
a target probe could be carried out when circularized probes
EHFRPHDYDLODEOH7KHVHWZRVWULFWFRQGLWLRQVFUHDWHDQLGHDO GHWHFWLRQ SODWIRUP IRU KLJKO\ VHQVLWLYH DQG VSHFL¿F 613V GHWHFWLRQ %\ LQFUHDVLQJ WKH K\EULGL]DWLRQ WHPSHUDWXUH DQG VKRUWHQLQJ WKH ¶ DUP EHORZ WKH UHDFWLRQ WHPSHUDWXUH WKH GLVFULPLQDWLRQ RI 613 FDQ EH IXUWKHU LPSURYHG 3LFNHULQJet al,)DUXTL et al. 7KLVPHWKRGIRU613VGHWHFWLRQ EDVHGRQ'1$OLJDVHPHGLDWHGVLQJOHQXFOHRWLGHGLVFULPLQDWLRQ ZLWK FRQVHFXWLYH VLJQDO DPSOL¿FDWLRQ E\ +5&$ KDV EHHQ
developed for various groups of pathogenic organisms,
LQFOXGLQJIXQJLEDFWHULDDQGYLUXVHV.RQJet al=KRX et al.DRFKDURHQet al.:DQJet al). 5HFHQWO\WKHWHFKQRORJ\KDVEHHQXVHGWRdifferentiate and to detect two closely related conifer pathogens vectored by the
PRXQWDLQSLQHEHHWOHV7VXLet al.
applications for clinical diagnosis as well as surveillance of infectious diseases in developing countries without requiring
VRSKLVWLFDWHGHTXLSPHQWRUVNLOOHGSHUVRQQHO0RUL 1RWRPL , 3DULGDet al.
7KHWHFKQLTXHZDV¿UVWGHVFULEHGDQGLQLWLDOO\HYDOXDWHG IRU GHWHFWLRQ RI KHSDWLWLV % YLUXV '1$ E\ 1RWDPLet al 1RWRPLet al. /$03DVVD\VKDYHEHHQPRVWO\XVHG IRU WKH GLDJQRVWLFV RI EDFWHULD &KHQet al. +DQet al. YLUXV :DQJet al. =KDRet al. DQG SDUDVLWHV ,NDGDLet al. ,VHNLet al. EXW LW KDV
also been developed for the rapid detection of pathogenic
RUDOOHUJHQLFIXQJL2KRULet alXVHGWKHWHFKQLTXHIRU UDSLG LGHQWL¿FDWLRQ RIOchroconis gallopava, an emerging fungal pathogen and causative agent of zoonotic infections,
ZKLOH6XQDQGFRZRUNHUVXVHGLWIRUWKHUDSLGGLDJQRVLVRI Penicillium marneffeiLQDUFKLYHGWLVVXHVDPSOHV6XQet al. DDQGRIFonsecaea agents of chromoblastomycosis
6XQet al. E6LPLODUO\(QGRet alDQG7DWLEDQD et al. GHWHFWHG WKH SUHVHQFH RI WKHgp43 gene in
Paracoccidioides brasiliensis E\ /$03 DQG /XFDVet al XVHG /$03 IRU GLIIHUHQWLDWLRQ RI Cryptococcus neoformans varieties from C. gattii EDVHG RQ &$3 VHTXHQFHV5HFHQWO\/Xet al.XWLOL]HGWKHWHFKQRORJ\ IRUWKHLGHQWL¿FDWLRQRIPseudallescheria and Scedosporium VSHFLHV
7ZRLQQHUDQGWZRRXWHUSULPHUVDUHUHTXLUHGIRU/$03 )LJ $ ,Q WKH LQLWLDO VWHSV RI WKH /$03 UHDFWLRQ DOO IRXU
primers are employed, but in the later cycling steps only
WKH LQQHU SULPHUV DUH XVHG IRU VWUDQG GLVSODFHPHQW '1$ V\QWKHVLV7KHRXWHUSULPHUVDUHUHIHUUHGWRDV)DQG% ZKLOH WKH LQQHU SULPHUV DUH IRUZDUG LQQHU SULPHU ),% DQG EDFNZDUGLQQHUSULPHU%,3%RWK),3DQG%,3FRQWDLQVWZR
distinct sequences corresponding to the sense and antisense
VHTXHQFHV RI WKH WDUJHW '1$ RQH IRU SULPLQJ LQ WKH ¿UVW VWDJHDQGWKHRWKHUIRUVHOISULPLQJLQODWHUVWDJHV1RWRPLet al.7KHVL]HDQGVHTXHQFHRIWKHSULPHUVZDVFKRVHQ VRWKDWWKHLUPHOWLQJWHPSHUDWXUH7PLVEHWZHHQ&
the optimal temperature for Bst SRO\PHUDVH 7KH )F DQG %F7PYDOXHVVKRXOGEHDOLWWOHKLJKHUWKDQWKRVHRI)DQG % WR IRUP WKH ORRSHGRXW VWUXFWXUH 7KH 7P YDOXHV RI WKH RXWHUSULPHUV)DQG%KDYHWREHORZHUWKDQWKRVHRI) DQG%WRDVVXUHWKDWWKHLQQHUSULPHUVVWDUWV\QWKHVLVHDUOLHU WKDQWKHRXWHUSULPHUV$GGLWLRQDOO\WKHFRQFHQWUDWLRQVRIWKH
inner primers are higher than the concentrations of the outer
SULPHUV1RWRPLet al.7RPLWDet al.)XUWKHUPRUH LWLVFULWLFDOIRU/$03WRIRUPDVWHPORRS'1$IURPDGXPE
Fig. 6. 6FKHPDWLF UHSUHVHQWDWLRQ RI WKH PHFKDQLVP RI /$03A.
*HQHUDOORFDWLRQRIWKH/$03SULPHUVHWLQUHODWLRQWRGH¿QHGUHJLRQV RI WKH WDUJHW '1$ )RUZDUG ) DQG EDFNZDUG % RXWHU SULPHUV DQG IRUZDUG ),3 DQG EDFNZDUG %,3 LQQHU SULPHUV DUH LQGLFDWHG
AR
TICLE
EHOO VWUXFWXUH 9DULRXV VL]HV RI WKH ORRS EHWZHHQ )F DQG )FDQGEHWZHHQ%FDQG%FZHUHH[DPLQHGDQGWKHEHVW UHVXOWV DUH REWDLQHG ZKHQ ORRSV RI QXFOHRWLGHV QW RU ORQJHU DUH XVHG 1RWRPLet al. 7KH VL]H RI WDUJHW '1$ LV DQ LPSRUWDQW IDFWRU WKDW /$03 HI¿FLHQF\ GHSHQGV RQEHFDXVHWKHUDWHOLPLWLQJVWHSIRUDPSOL¿FDWLRQLVVWUDQG GLVSODFHPHQW'1$V\QWKHVLV
/$03 UHOLHV RQ DXWRF\FOLQJ VWUDQG GLVSODFHPHQW '1$
synthesis in the presence of Bst'1$SRO\PHUDVHVSHFL¿F SULPHUV DQG WKH WDUJHW '1$ WHPSODWH 7KH PHFKDQLVP RI WKH /$03 DPSOL¿FDWLRQ UHDFWLRQ LQFOXGHV WKUHH VWHSV SURGXFWLRQ RI VWDUWLQJ PDWHULDO F\FOLQJ DPSOL¿FDWLRQ DQG UHF\FOLQJ1RWRPLet al.7RPLWDet al.)LJ%& 7RSURGXFHWKHVWDUWLQJPDWHULDOLQQHUSULPHU),%K\EULGL]HV WR)FLQWKHWDUJHW'1$DQGLQLWLDWHVFRPSOHPHQWDU\VWUDQG V\QWKHVLV 2XWHU SULPHU ) K\EULGL]HV WR )F LQ WKH WDUJHW DQGLQLWLDWHVVWUDQGGLVSODFHPHQW'1$V\QWKHVLVUHOHDVLQJ D),3OLQNHGFRPSOHPHQWDU\VWUDQGZKLFKIRUPVDORRSHG RXWVWUXFWXUHDWRQHHQG'1$DPSOL¿FDWLRQSURFHHGVZLWK %,3LQDVLPLODUPDQQHU7KLVVLQJOHVWUDQGHG'1$VHUYHV DVWHPSODWHIRU%,3LQLWLDWHG'1$V\QWKHVLVDQGVXEVHTXHQW %SULPHG VWUDQG GLVSODFHPHQW '1$ V\QWKHVLV OHDGLQJ WR WKH SURGXFWLRQ RI D GXPEEHOO IRUP '1$ ZKLFK LV TXLFNO\ FRQYHUWHG WR D VWHP ORRS '1$ 7KLV WKHQ VHUYHV DV WKH VWDUWLQJPDWHULDOIRU/$03F\FOLQJWKHVHFRQGVWDJHRIWKH /$03UHDFWLRQ'XULQJF\FOLQJDPSOL¿FDWLRQ),3K\EULGL]HV WR WKH ORRS LQ WKH VWHPORRS '1$ DQG SULPHV VWUDQG GLVSODFHPHQW'1$V\QWKHVLVJHQHUDWLQJDVDQLQWHUPHGLDWH RQHJDSSHGVWHPORRS'1$ZLWKDQDGGLWLRQDOLQYHUWHGFRS\
of the target sequence in the stem, and a loop formed at
WKH RSSRVLWH HQG YLD WKH %,3 VHTXHQFH 6XEVHTXHQW VHOI SULPHG VWUDQG GLVSODFHPHQW '1$ V\QWKHVLV \LHOGV RQH FRPSOHPHQWDU\VWUXFWXUHRIWKHRULJLQDOVWHPORRS'1$DQG RQHJDSUHSDLUHGVWHPORRS'1$ZLWKDVWHPHORQJDWHGWR WZLFHDVORQJDQGDORRSDWWKHRSSRVLWHHQG%RWKRIWKHVH SURGXFWV WKHQ VHUYH DV WHPSODWHV IRU %,3SULPHG VWUDQG
displacement in the subsequent cycles, the elongation and
UHF\FOLQJVWHS7KH¿QDOSURGXFWLVDPL[WXUHRIVWHPORRS '1$ZLWKYDULRXVVWHPOHQJWKDQGFDXOLÀRZHUOLNHVWUXFWXUHV
with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand
1RWRPL et al.7RPLWDet al.
/$03SURGXFWVFDQEHGLUHFWO\REVHUYHGE\WKHQDNHGH\H
or using a UV transilluminator in the reaction tube by adding
ORIIROGGLOXWHG6<%5*UHHQ,VWDLQWRWKHUHDFWLRQ WXEH VHSDUDWHO\ 8QGHU 89 LOOXPLQDWLRQ WKH JHO VKRZV D ODGGHUOLNHVWUXFWXUHIURPWKHPLQLPXPOHQJWKRIWDUJHW'1$
up to the loading well, which are the various length stem-loop
SURGXFWVRIWKH/$03UHDFWLRQ
CONCLUSION
1XPHURXV GHWHFWLRQ PHWKRGRORJLHV DUH QRZ DYDLODEOH EXW
regardless of the approach, important questions need to be
DQVZHUHG SULRU WR WKHLU LQFOXVLRQ LQWR H[SHULPHQWV 7KHVH
include sensitivity, accuracy, robustness, frequency of testing,
DQGFRVW'HVSLWHPDQ\QRYHOWHFKQRORJLHVEHLQJDYDLODEOH
challenges remain to identify as yet unculturable fungi, to detect cryptic species, and to characterize the assemblage and diversity of fungal communities in different environments
ZLWKRXW ELDV 7KHUH LV DOZD\V D QHHG WR FKDUDFWHUL]H IXQJL TXLFNO\ DQG DFFXUDWHO\ 1R RQH NQRZV KRZ PDQ\ IXQJDO VSHFLHV H[LVW EXW VHTXHQFLQJ RI HQYLURQPHQWDO '1$ PD\ LPSURYH WKH DFFXUDF\ RI FXUUHQW HVWLPDWHV +DZNVZRUWK 1H[WJHQHUDWLRQ VHTXHQFLQJ DQG S\URVHTXHQFLQJ
approaches will also provide promising ways of enlarging the scope of molecular-detection studies
ACKNOWLEDGEMENTS
:HZRXOGOLNHWRWKDQNWKH,0&RUJDQL]HUVIRUVXSSRUWLQJWKLV6,* V\PSRVLXP:HDUHWKDQNIXOWR6DWRNR<DPDPRWR(LNHQ&KHPLFDO -DSDQIRUSHUPLVVLRQWRXVHGLDJUDPVRI/$03IURPKWWSORRSDPS HLNHQFRMSHODPSSULQFLSOHKWPO!
REFERENCES
$JLQGRWDQ % 3HUU\ ./ 0DFURDUUD\ GHWHFWLRQ RI SODQW 51$ YLUXVHV XVLQJ UDQGRPO\ SULPHG DQG DPSOL¿HG FRPSOHPHQWDU\ '1$VIURPLQIHFWHGSODQWVPhytopathology97± $JLQGRWDQ % 3HUU\ ./ 0DFURDUUD\ GHWHFWLRQ RI HOHYHQ
SRWDWRLQIHFWLQJ YLUXVHV DQG 3RWDWR VSLQGOH WXEHU YLURLGPlant
Disease92±
$JULRV*Plant PathologythHGQ/RQGRQ(OVHYLHU$FDGHPLF
3UHVV
$PDQQ 5, /XGZLJ : DQG 6FKOHLIHU .+ 3K\ORJHQHWLF LGHQWL¿FDWLRQ DQGin situ detection of individual microbial cells
ZLWKRXWFXOWLYDWLRQMicrobiology and Molecular Biology Reviews
59±
%DNHU %- /XW] 0$ 'DZVRQ 6& %RQG 3/ %DQ¿HOG -) 0HWDEROLFDOO\DFWLYHHXNDU\RWLFFRPPXQLWLHVLQH[WUHPHO\DFLGLF PLQH GUDLQDJHApplied and Environmental Microbiology 70:
±
%DQpU-1LOVVRQ00HQGHO+DUWYLJ0/DQGHJUHQ86LJQDO DPSOL¿FDWLRQ RI SDGORFN SUREHV E\ UROOLQJ FLUFOH UHSOLFDWLRQ
Nucleic Acids Research26±
%DVFKLHQ&0DQ]:1HX756]HZ]\N8)OXRUHVFHQFHin situ K\EULGL]DWLRQ RI IUHVKZDWHU IXQJLInternational Review of Hydrobiology86±
%DVFKLHQ&0DQ]:1HX750DUYDQRYi/6]HZ]\N8In situ detection of freshwater fungi in an alpine stream by new
WD[RQVSHFL¿FÀXRUHVFHQFHin situ hybridization probes.Applied and Environmental Microbiology74±
%ULHUOH\-/6WHZDUW-$/HHV$.4XDQWLI\LQJSRWDWRSDWKRJHQ '1$LQVRLOApplied Soil Ecology±
%UXO61XVVEDXP-'LHOEDQGKRHVLQJ6.)OXRUHVFHQWSUREHV IRUFHOOZDOO SRURVLW\DQGPHPEUDQH LQWHJULW\LQ¿ODPHQWRXV IXQJLJournal of Microbiological Methods28±
&KHKDE )) -HII : 'HWHFWLRQ RI PXOWLSOH F\VWLF ¿EURVLV
mutations by reverse dot blot hybridization: a technology for
DPSOL¿FDWLRQ /$03 PHWKRGFEMS Microbiology Letters234:
±
(ULNVVRQ 5 -REV 0 (NVWUDQG & 8OOEHUJ 0 +HUUPDQQ % /DQGHJUHQ 8 1LOVVRQ 0 %ORPEHUJ - 0XOWLSOH[ DQG TXDQWL¿DEOH GHWHFWLRQ RI QXFOHLF DFLG IURP SDWKRJHQLF IXQJL XVLQJ SDGORFN SUREHV JHQHULF UHDO WLPH 3&5 DQG VSHFL¿F VXVSHQVLRQDUUD\UHDGRXWJournal of Microbiological Methods
78±
)DUXTL$) +RVRQR 6 'ULVFROO 0' 'HDQ )%$OVPDGL 2 %DQGDUX 5.XPDU**ULPZDGH%=RQJ46XQ='X<.LQJVPRUH6 .QRWW7/DVNHQ56+LJKWKURXJKSXWJHQRW\SLQJRIVLQJOH QXFOHRWLGHSRO\PRUSKLVPVZLWKUROOLQJFLUFOHDPSOL¿FDWLRQBMC Genomics2
)HVVHKDLH$'H%RHU6+/pYHVTXH&$$QROLJRQXFOHRWLGH DUUD\ IRU WKH LGHQWL¿FDWLRQ DQG GLIIHUHQWLDWLRQ RI EDFWHULD SDWKRJHQLFRQSRWDWRPhytopathology93±
)LUH $ ;X 64 5ROOLQJ UHSOLFDWLRQ RI VKRUW '1$ FLUFOHV
Proceedings of the National Academy of Sciences, USA 92:
±
*LOEHUW&$=KDQJ1+XWPDFKHU5%'DYLV506PDUW&' 'HYHORSPHQWRID'1$EDVHGPDFURDUUD\IRUWKHGHWHFWLRQDQG LGHQWL¿FDWLRQRIFusarium oxysporumIVSYDVLQIHFWXPLQFRWWRQ WLVVXHJournal of Cotton Science12±
+DQ +- -XQJ 6- 2K 0- .LP '+ 5DSLG DQG VHQVLWLYH
detection of Streptococcus iniae by loop-mediated isothermal
DPSOL¿FDWLRQ/$03Journal of Fish Disease34± +DUSHU .$ 6PDUW &' 0LFKDHO 'DYLV 5 'HYHORSPHQW RI D
'1$EDVHG PDFURDUUD\ IRU WKH GHWHFWLRQ DQG LGHQWL¿FDWLRQ RI
AmanitaVSHFLHVJournal of Forensic Science56± +DZNVZRUWK '/ 7KH IXQJDO GLPHQVLRQ RI ELRGLYHUVLW\
PDJQLWXGH VLJQL¿FDQFH DQG FRQVHUYDWLRQ Mycological Research95±
+DZNVZRUWK '/ 7KH PDJQLWXGH RI IXQJDO GLYHUVLW\ WKH PLOOLRQ VSHFLHV HVWLPDWH UHYLVWHGMycological Research 105:
±
,NDGDL + 7DQDND + 6KLEDKDUD 1 0DWVXX $ 8HFKL 0 ,WRK 1 2VKLUR 6 .XGR 1 ,JDUDVKL , 2\DPDGD 7 0ROHFXODU
evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated
LVRWKHUPDODPSOL¿FDWLRQPHWKRGJournal of Clinical Microbiology
42±
,QiFLR-%HKUHQV6)XFKV%0)RQVHFDÈ6SHQFHU0DUWLQV,$PDQQ 5In situ accessibility of Saccharomyces cerevisiae 6 U51$ WR F\ODEHOHG ROLJRQXFOHRWLGH SUREHV FRPSULVLQJ WKH ' DQG ' GRPDLQVApplied and Environmental Microbiology69:
±
FRPSOH[Electrophoresis29±
.DZDVDNL ( 6DLNL 5 (UOLFK + *HQHWLF DQDO\VLV XVLQJ SRO\PHUDVH FKDLQ UHDFWLRQDPSOL¿HG '1$ DQG LPPRELOL]HG ROLJRQXFOHRWLGH SUREHV UHYHUVH GRWEORW W\SLQJMethods in Enzymology 218±
.RQJ )7RQJ = &KHQ ; 6RUUHOO 7 :DQJ % :X 4 (OOLV ' &KHQ 6&5DSLGLGHQWL¿FDWLRQDQGGLIIHUHQWLDWLRQRITrichophyton
species, based on sequence polymorphisms of the ribosomal
LQWHUQDOWUDQVFULEHGVSDFHUUHJLRQVE\UROOLQJFLUFOHDPSOL¿FDWLRQ
Journal of Clinical Microbiology46±
/DQGHJUHQ8.DLVHU56DQGHUV-+RRG/$OLJDVHPHGLDWHG JHQHGHWHFWLRQWHFKQLTXHScience241±
/DX$&KHQ66OHLPDQ66RUUHOO7&XUUHQWVWDWXVDQGIXWXUH
perspectives on molecular and serological methods in diagnostic
P\FRORJ\Future Microbiology4±
/DX$6RUUHOO7&&KHQ66WDQOH\.,UHGHOO-+DOOLGD\&D 0XOWLSOH[WDQGHP3&5DQRYHOSODWIRUPIRUUDSLGGHWHFWLRQDQG LGHQWL¿FDWLRQRIIXQJDOSDWKRJHQVIURPEORRGFXOWXUHVSHFLPHQV
Journal of Clinical Microbiology46±
/DX $ 6RUUHOO 7& /HH 2 6WDQOH\ . +DOOLGD\ & E &RORQ\ PXOWLSOH[WDQGHP3&5IRUUDSLGDFFXUDWHLGHQWL¿FDWLRQRIIXQJDO FXOWXUHVJournal of Clinical Microbiology46± /DX$ +DOOLGD\ & &KHQ 6&$ 3OD\IRUG (* 6WDQOH\ . 6RUUHOO7&
&RPSDULVRQRIZKROHEORRGVHUXPDQGSODVPDIRUHDUO\ GHWHFWLRQ RI FDQGLGHPLD E\ PXOWLSOH[WDQGHP 3&5Journal of Clinical Microbiology48±
/H )ORFK * 7DPERQJ - 9DOODQFH - 7LULOO\ < /pYHVTXH $ 5H\ 3 5KL]RVSKHUH SHUVLVWHQFH RI WKUHHPythium oligandrum
VWUDLQVLQWRPDWRVRLOOHVVFXOWXUHDVVHVVHGE\'1$PDFURDUUD\ DQGUHDOWLPH3&5FEMS Microbiology Ecology61± /pYHVTXH &$ +DUOWRQ &( GH &RFN $:$0 ,GHQWL¿FDWLRQ
RI VRPH RRP\FHWHV E\ UHYHUVH GRW EORW K\EULGL]DWLRQ
Phytopathology 88±
/L6&XOOHQ'+MRUW06SHDU5$QGUHZV-+'HYHORSPHQW
of an oligonucleotide probe for Aureobasidium pullulans based
RQ WKH VPDOO VXEXQLW U51$ JHQHApplied and Environmental Microbiology62±
/LHYHQV%%URXZHU09DQDFKWHU$&5&/pYHVTXH&$&DPPXH%3$ 7KRPPD%3+-'HVLJQDQGGHYHORSPHQWRID'1$DUUD\ IRU UDSLG GHWHFWLRQ DQG LGHQWL¿FDWLRQ RI PXOWLSOH WRPDWR YDVFXODU ZLOWSDWKRJHQVFEMS Microbiology Letters223± /LHYHQV%&ODHV/9DQDFKWHU$&&DPPXH%37KRPPD%3
'HWHFWLQJ VLQJOH QXFOHRWLGH SRO\PRUSKLVPV XVLQJ '1$ DUUD\V IRU SODQW SDWKRJHQ GLDJQRVLVFEMS Microbiology Letters255:
AR
TICLE
/L]DUGL30+XDQJ;=KX=%UD\:DUG37KRPDV'&:DUG'& 0XWDWLRQ GHWHFWLRQ DQG VLQJOHPROHFXOH FRXQWLQJ XVLQJ LVRWKHUPDOUROOLQJFLUFOHDPSOL¿FDWLRQNature Genetics19±
/X 4 *HUULWV YDQ GHQ (QGH$+ %DNNHUV -0 6XQ - /DFNQHU 0 1DMDI]DGHK 0- 0HOFKHUV :- /L 5 GH +RRJ *6 ,GHQWL¿FDWLRQ RIPseudallescheria and Scedosporium species
E\WKUHHPROHFXODUPHWKRGVJournal of Clinical Microbiology49:
/XFDV 6 GD /X] 0DUWLQV 0 )ORUHV 2 0H\HU : 6SHQFHU0DUWLQV , ,QiFLR - 'LIIHUHQWLDWLRQ RICryptococcus neoformans
varieties and Cryptococcus gattii XVLQJ &$3EDVHG ORRS PHGLDWHG LVRWKHUPDO '1$ DPSOL¿FDWLRQClinical Microbiology and Infection16±
0F$UWKXU )$ %lUORFKHU 02 0F/HDQ 1$% +LOW] 0' %lUORFKHU ) $VNLQJ SURELQJ TXHVWLRQV FDQ ÀXRUHVFHQW LQ VLWX
hybridization identify and localize aquatic hyphomycetes on leaf litter? International Review of Hydrobiology86 0RUL < 1RWRPL 7 /RRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQ
/$03DUDSLGDFFXUDWHDQGFRVWHIIHFWLYHGLDJQRVWLFPHWKRG IRULQIHFWLRXVGLVHDVHVJournal of Infection and Chemotherapy
15±
1LOVVRQ 0 0DOPJUHQ + 6DPLRWDNL 0 .ZLDWNRZVNL 0 &KRZGKDU\ %3 /DQGHJUHQ 8 3DGORFN SUREHV FLUFXODUL]LQJ ROLJRQXFOHRWLGHV IRU ORFDOL]HG '1$ GHWHFWLRQScience 265:
±
1LOVVRQ 0 .UHMFL . .RFK - .ZLDWNRZVNL 0 *XVWDYVVRQ 3 /DQGHJUHQ 8 3DGORFN SUREHV UHYHDO VLQJOHQXFOHRWLGH
differences, parent of origin and in situ distribution of centromeric
VHTXHQFHVLQKXPDQFKURPRVRPHVDQGNature Genetics
16±
1LOVVRQ 0 'DKO ) /DUVVRQ & *XOOEHUJ 0 6WHQEHUJ - $QDO\]LQJJHQHVXVLQJFORVLQJDQGUHSOLFDWLQJFLUFOHVTrends in Biotechnology24±
1RWRPL 7 2ND\DPD + 0DVXEXFKL + <RQHNDZD 7 :DWDQDEH . $PLQR1+DVH7/RRSPHGLDWHGLVRWKHUPDODPSOL¿FDWLRQ RI'1$Nucleic Acids Research28(
2KRUL$(QGR66DQR$<RNR\DPD.<DULWD.<DPDJXFKL0.DPHL .0L\DML01LVKLPXUD.5DSLGLGHQWL¿FDWLRQRIOchroconis gallopava E\ D ORRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQ /$03 PHWKRGVeterinary Microbiology114±
2NXEDUD3$6FKURHGHU.//L&6FKXPDFKHU57/DZUHQFH13 ,PSURYHGH[WUDFWLRQRIRhizoctonia and Pythium'1$
from wheat roots and soil samples using pressure cycling
WHFKQRORJ\Canadian Journal of Plant Pathology 29 ±
2SKHO.HOOHU . 0F.D\$ +DUWOH\ ' +HUGLQD$ &XUUDQ - 'HYHORSPHQW RI D URXWLQH '1$EDVHG WHVWLQJ VHUYLFH IRU VRLOERUQH GLVHDVHV LQ $XVWUDOLDAustralasian Plant Pathology
37±
3DULGD06DQQDUDQJDLDK6'DVK3.5DR390RULWD./RRS PHGLDWHGLVRWKHUPDODPSOL¿FDWLRQ/$03DQHZJHQHUDWLRQRI LQQRYDWLYHJHQHDPSOL¿FDWLRQWHFKQLTXHSHUVSHFWLYHVLQFOLQLFDO GLDJQRVLVRILQIHFWLRXVGLVHDVHVReviews in Medical Virology18:
±
3HUQWKDOHU $ 3HUQWKDOHU - $PDQQ 5 )OXRUHVFHQFHin situ hybridization and catalyzed reporter deposition for the
LGHQWL¿FDWLRQ RI PDULQH EDFWHULDApplied and Environmental Microbiology68±
3LFNHULQJ-%DPIRUG$*RGEROH9%ULJJV-6FR]]DIDYD*5RH3 :KHHOHU&*KRX]H)&XVV6,QWHJUDWLRQRI'1$OLJDWLRQ DQG UROOLQJ FLUFOH DPSOL¿FDWLRQ IRU WKH KRPRJHQHRXV HQGSRLQW GHWHFWLRQ RI VLQJOH QXFOHRWLGH SRO\PRUSKLVPVNucleic Acids Research30H
3R]KLWNRY$1REOH3$'RPD]HW/RVßR71ROWH$:6RQQHQEHUJ5 6WDHKOHU3%HLHU07DXW]'7HVWVRIU51$K\EULGL]DWLRQ
to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be
SUHGLFWHGNucleic Acids Research 34H
3R]KLWNRY$(7DXW]'1REOH3$2OLJRQXFOHRWLGHPLFURDUUD\V ZLGHO\ DSSOLHG ± SRRUO\ XQGHUVWRRG%ULH¿QJV LQ )XQFWLRQDO Genomics & Proteomics6±
3ULQV7:YDQ'LMN-3%HHQHQ+*9DQ+RHI$$9RRUKXLM]HQ00 6FKRHQ&'$DUWV+-.RN(-2SWLPLVHGSDGORFNSUREH OLJDWLRQ DQG PLFURDUUD\ GHWHFWLRQ RI PXOWLSOH QRQDXWKRULVHG *02VLQDVLQJOHUHDFWLRQBMC Genomics 9
3XQMD=.:DQ$*RVZDPL569HUPD15DKPDQ0%DUDVXEL\H 76HLIHUW.$/pYHVTXH&$'LYHUVLW\RIFusarium species
DVVRFLDWHG ZLWK GLVFRORUHG JLQVHQJ URRWV LQ %ULWLVK &ROXPELD
Canadian Journal of Plant Pathology29±
5HHOHGHU 5' &DSHOOD %% 7RPOLQVRQ /' +LFNH\ :- 7KH H[WUDFWLRQ RI IXQJDO '1$ IURP PXOWLSOH ODUJH VRLO VDPSOHV
Canadian Journal of Plant Pathology25±
5RELGHDX*3&DUXVR)/2XGHPDQV390F0DQXV365HQDXG0$ $XFODLU0(%LORGHDX*-<HH''pVDXOQLHUV1/'H9HUQD-: /pYHVTXH&$'HWHFWLRQRIFUDQEHUU\IUXLWURWIXQJLXVLQJ '1$ DUUD\ K\EULGL]DWLRQCanadian Journal of Plant Pathology
30±
5RELQ%-$UIID5&$YQL,5DR1$5DSLGYLVXDOL]DWLRQRIWKUHH FRPPRQIXQJLXVLQJÀXRUHVFHLQFRQMXJDWHGOHFWLQVInvestigative Ophthalmology & Visual Science27±
6FXSKDP $- 3UHVOH\ // :HL % %HQW ( *ULI¿WK 1 0F3KHUVRQ 0 =KX ) 2OXZDGDUD 2 5DR 1 %UDXQ - %RUQHPDQ - $EXQGDQWDQGGLYHUVHIXQJDOPLFURELRWDLQWKHPXULQHLQWHVWLQH
Applied and Environmental Microbiology72±
6HLIHUW.$/pYHVTXH&$3K\ORJHQ\DQGPROHFXODUGLDJQRVLV RI P\FRWR[LJHQLF IXQJLEuropean Journal of Plant Pathology
110±
6HLIHUW.$6DPVRQ5$GH:DDUG-5+RXEUDNHQ-/pYHVTXH&$ 0RQFDOYR-0/RXLV6HL]H*DQG+HEHUW3'13URVSHFWV IRU IXQJXV LGHQWL¿FDWLRQ XVLQJ &2 '1$ EDUFRGHV ZLWK
PenicilliumDVDWHVWFDVHProceedings of the National Academy of Sciences, USA 104±
6KHSDUG -5 $GGLVRQ 50 $OH[DQGHU %' 'HOOD/DWWD 3 *KHUQD 0 +DDVH * +DOO * -RKQVRQ -. 0HU] :* 3HOWURFKH /ODFVDKXDQJD+6WHQGHU+9HQH]LD5$:LOVRQ'3URFRS*: :X))LDQGDFD0-0XOWLFHQWHUHYDOXDWLRQRIWKHCandida albicansCandida glabrataSHSWLGHQXFOHLFDFLGÀXRUHVFHQWLQVLWX K\EULGL]DWLRQPHWKRGIRUVLPXOWDQHRXVGXDOFRORULGHQWL¿FDWLRQRI
C. albicans and C. glabrata GLUHFWO\ IURP EORRG FXOWXUH ERWWOHV
Journal of Clinical Microbiology46±
'HYHORSPHQW DQG HYDOXDWLRQ RI ORRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQ /$03 IRU WKH UDSLG GLDJQRVLV RIPenicillium marneffei LQ DUFKLYHG WLVVXH VDPSOHVFEMS Immunology and Medical Microbiology58±
6XQ - 1DMDI]DGHK 0- 9LFHQWH 9 ;L / GH +RRJ *6 E 5DSLG GHWHFWLRQ RI SDWKRJHQLF IXQJL XVLQJ ORRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQ H[HPSOL¿HG E\Fonsecaea agents of
FKURPREODVWRP\FRVLVJournal of Microbiological Methods80:
±
6]HPHV0%RQDQWV3GH:HHUGW0%DQHU-/DQGHJUHQ86FKRHQ &''LDJQRVWLFDSSOLFDWLRQRISDGORFNSUREHV±PXOWLSOH[ GHWHFWLRQRISODQWSDWKRJHQVXVLQJXQLYHUVDOPLFURDUUD\VNucleic Acids Research 33H
7DPERQJ -7 GH &RFN $:$0 7LQNHU 1$ /pYHVTXH &$ 2OLJRQXFOHRWLGHDUUD\IRULGHQWL¿FDWLRQDQGGHWHFWLRQRIPythium
VSHFLHVApplied and Environmental Microbiology 72 ±
7DWLEDQD%76DQR$8QR-.DPHL.,JDUDVKL70LNDPL<0L\DML 01LVKLPXUD.,WDQR(1'HWHFWLRQRIParacoccidioides brasiliensis JS JHQHin sputa by loop-mediated isothermal
DPSOL¿FDWLRQPHWKRGJournal of Clinical Laboratory Analysis23:
±
7RPLWD 1 0RUL < .DQGD + 1RWRPL 7 /RRSPHGLDWHG LVRWKHUPDODPSOL¿FDWLRQ/$03RIJHQHVHTXHQFHVDQGVLPSOH YLVXDOGHWHFWLRQRISURGXFWVNature Protocols3± 7VXL &.0 :DQJ % .KDGHPSRXU / $ODPRXWL 60 %RKOPDQQ
- 0XUUD\ %: +DPHOLQ 5& 5DSLG LGHQWL¿FDWLRQ DQG
detection of pine pathogenic fungi associated with mountain pine
EHHWOHVE\SDGORFNSUREHVJournal of Microbiological Methods
83±
7VXL&.0:DQJ%6FKRHQ&+DPHOLQ5&3DGORFNSUREH IRU WKH GHWHFWLRQ RI SDWKRJHQLF IXQJL ,QLaboratory Protocols in Fungal Biology: Current Methods in Fungal Biology *XSWD 9$7XRK\0$\\DFKDP\07XUQHU.02¶'RQRYDQ$HGVLQ SUHVV6SULQJHU9HUODJ
8HKDUD 7 .XVKLGD $ 0RPRWD < 5DSLG DQG VHQVLWLYH LGHQWL¿FDWLRQ RI Pratylenchus VSS XVLQJ UHYHUVH GRW EORW K\EULGL]DWLRQNematology 1±
8UDNDZD + )DQWURXVVL 6( 6PLGW + 6PRRW -& 7ULERX (+ .HOO\ --1REOH3$6WDKO'$2SWLPL]DWLRQRIVLQJOHEDVHSDLU PLVPDWFKGLVFULPLQDWLRQLQROLJRQXFOHRWLGHPLFURDUUD\VApplied and Environmntal Microbiology69±
YDQ 'RRUQ 5 6]HPHV 0 %RQDQWV 3 .RZDOFKXN *$ 6DOOHV -) 2UWHQEHUJ(6FKRHQ&'4XDQWLWDWLYHPXOWLSOH[GHWHFWLRQ
of plant pathogens using a novel ligation probe-based system
FRXSOHG ZLWK XQLYHUVDO KLJKWKURXJKSXW UHDOWLPH 3&5 RQ
FLUFOHDPSOL¿FDWLRQJournal of Virological Methods161± :DQJ % 'Z\HU '( %O\WK && 6RHGMRQR 0 6KL + .HVVRQ $
5DWQDPRKDQ00F3KLH.&XQQLQJKDP$/6DNVHQD1. 'HWHFWLRQRIWKHUDSLGHPHUJHQFHRIWKH+<PXWDWLRQDVVRFLDWHG ZLWKRVHOWDPLYLUUHVLVWDQFHLQVHYHUHSDQGHPLFLQÀXHQ]DYLUXV$ +1LQIHFWLRQVAntiviral Research87±
:DQJ<.DQJ=*DR+*DR<4LQ//LQ+<X)4L;:DQJ; $RQHVWHSUHYHUVHWUDQVFULSWLRQORRSPHGLDWHGLVRWKHUPDO DPSOL¿FDWLRQIRUGHWHFWLRQDQGGLVFULPLQDWLRQRILQIHFWLRXVEXUVDO GLVHDVHYLUXVVirology Journal8
:HEHU 6' /XGZLJ : 6FKOHLIHU .+ )ULHG - 0LFURELDO FRPSRVLWLRQDQGVWUXFWXUHRIDHURELFJUDQXODUVHZDJHELR¿OPV.
Applied and Environmental Microbiology73± :LOVRQ '$ -R\FH 0- +DOO /6 5HOOHU /% 5REHUWV *' +DOO *6
$OH[DQGHU %' 3URFRS : 0XOWLFHQWHU HYDOXDWLRQ RI
a Candida albicans SHSWLGH QXFOHLF DFLG ÀXRUHVFHQW LQ VLWX
hybridization probe for characterization of yeast isolates from
EORRGFXOWXUHVJournal of Clinical Microbiology43± ;LRQJ/.RQJ)<DQJ<&KHQJ-*LOEHUW*/8VHRI3&5DQG
UHYHUVH OLQH EORW K\EULGL]DWLRQ PDFURDUUD\ EDVHG RQ 66 U51$ JHQH LQWHUQDO WUDQVFULEHG VSDFHU VHTXHQFHV IRU UDSLG LGHQWL¿FDWLRQ RI Mycobacterium VSHFLHVJournal of Clinical Microbiology44±
<DQJ <3 &RUOH\ 1 *DUFLD+HUDV - 5HYHUVH GRWEORW
hybridization as an improved tool for the molecular diagnosis of point mutations in congenital adrenal hyperplasia caused by
K\GUR[\ODVHGH¿FLHQF\Molecular Diagnostics 6± =KDQJ 1 *HLVHU 0' 6PDUW &' 0DFURDUUD\ GHWHFWLRQ RI
solanaceous plant pathogens in the Fusarium solani species
FRPSOH[Plant disease91±
=KDQJ10F&DUWK\0/6PDUW&'$PDFURDUUD\V\VWHPIRU
the detection of fungal and oomycete pathogens of solanaceous
FURSVPlant disease 92±
=KDQJ< &R\QH 0< :LOO 6* /HYHQVRQ &+ .DZDVDNL (6 6LQJOHEDVHPXWDWLRQDODQDO\VLVRIFDQFHUDQGJHQHWLFGLVHDVHV XVLQJPHPEUDQHERXQGPRGL¿HGROLJRQXFOHRWLGHVNucleic Acids
Research 19±
=KDR . 6KL : +DQ ) ;X < =KX / =RX < :X ; =KX + 7DQ ) 7DR 6 7DQJ ; 6SHFL¿F VLPSOH DQG UDSLG GHWHFWLRQ RI
porcine circovirus W\SH XVLQJ WKH ORRSPHGLDWHG LVRWKHUPDO DPSOL¿FDWLRQPHWKRGVirology Journal8
