f n ùôûãÿ e l¢ ¡] .6¡¨/§e - niid.go.jp · bsl2_ )o °= r 3] uxà ýt Ð>6 è k ¯`Êj...
TRANSCRIPT
2
jR�
�
���eÛO��
�
¡��NMÚ�Öá�viU4�
� ¡� ��'�(Ñ'�t�
� ¡�¡��NMÛ�ÞA�
� ¡�¢���eÛNMH?Ñ;�CF�
� ¡�£��NMH?Û��Øn�
� ¡�¤��NMÛ�&�
� ¡�¥��5LgT*Õ� ZÚÔÕáf���@Û�à7Ó�
�
¢��zX$iNM�
� ¢� ���z`��8��±¾Ã»¿¿ÂËÊÁÄ»ÊÁÅÄ�²ÄÀÁ¼ÁÊÁÅÄ©�±²�9�Y&T�
� � ¢� � ���w��
� � ¢� �¡��zX�a�
� � ¢� �¢��±¬ 9�Û&��
� � ¢� �£��±² G���
� � ¢� �¤���viÙU4�
� � ¢� �¥���Ûj%�
� ¢�¡��²¿³ �p d9�Y&� �²¿³�®²¬��
� � ¢�¡� ���Ûj%�
� ¢�¢��²¿° �p d9�Y&� �²¿°�®²¬��
� � ¢�¢� ���Ûj%�
�
£����#$iNM�
� £� ��¹»Ç³»Ä ÷āĄöâcÓ×þãÿîäú ·¹�¶· T�
� � £� � ���Ù�wăQ��
� � £� �¡��e�\ ·´¬ âcÓ×��ÛD���
� � £� �¡� ��e�\ ·´¬Û,��
� � £� �¡�¡��2VÛ�{�
� � £� �¡�¢� þãÿîäú ·¹�¶· �2�
3
� � £� �¡�£� ñĄî|J�
� � £� �¢�NMÛ'B�
� � £� �¢� �åäÿí ·´¬:��
� � £� �¢�¡��e�\·´¬Û�=�
� � £� �¢�¢��eåäÿíéõúÛN��
� � £� �¢�£���6kI��
� � £� �¢�¤� �&�
� � £� �¢�¥� òýöÿÝÛ)2�
� £�¡� êĂøĂìüóÿ ·¹�ľÉʾ½� ¶· Tċ�P�xh�ą´¸Ć��#"-
T�
� � £�¡� �¹ÍÅ�ÉÊ¾Æ TÚßá�eåäÿí ´¸��#ÛN��
� � � £�¡� � ��w�
� � � £�¡� �¡�åäÿí ·´¬:��
� � � £�¡� �¢������2ą·¹Ć�
� � � £�¡� �£�� ÉÊ�¶· ÔßÜ Ä¾Éʾ½�¶· �2�
� � £�¡�¡��µÄ¾�ÉʾÆ�·¹�¶· TÚßá�eåäÿí ´¸��#ÛN��
� � � £�¡�¡� ��w�
� � � £�¡�¡�¡�µÄ¾�ÉʾÆ�·¹�¶· ÔßÜ ´¾Éʾ½�¶· �2�
� £�¢���eåäÿí ® ��#ÛN�ÔßÜ��#|J�
� � £�¢� ���w�
� � £�¢�¡�� ÉÊ�¶· ÔßÜ Ä¾Éʾ½�¶·�
� � £�¢�¢��¶· b^Ûo{ØïäĀèòìĄèçĂí�2�
� � £�¢�£�� ìĄèçĂísKÛ|J�
� £�£�� þãÿîäú ·¹�¶· ÔßÜêĂøĂìüóÿ ·¹�¶· Û�3êĂòā
Ąÿ�
�
¤�� åäÿí���
� ¤� ���cqu�
� ¤�¡����AT�
� ¤�¢���&AT�
� � ¤�¢� ��y�9�T�
�
5
�� �eÛO� � ɡ` Togavirus ɼ Rubivirus Ž_£±� RNA zy³�Y<pɡz
y³�_ȘƩdz]mUXƒC˵IJqpơƢƩdzɤY<o5ƩdzƘ;
_ȸÚǥtʞXɦɤMp6ɦɄ38~39+ ¹ɦɡ÷˷Ƣ_Źʙǐl
ʙ˄Âɡ ¹²·�ʏʼʹÆ]ʰÒƘ5@mbƘB¼ÆƝY<
p6ɦɄ¹ɦɡ`ǍǗYȧŠMpB5²·�ʏʼʹ`;ƹʢMp6
ƭÑY`ʏȾ_ɤɋkKaKa˪jnqpB5eZu[`»Ƣ]ʝƣM
p6ʀ]ˎŹǯȭźƢʛǐɣlʸȾʊ_įáɤt˛pIZk<pB5ŒǨɪ
]ÊƘ`˃ţ\ɢƤY<p6gR¿ƢƩdzkŹõY 30~50%5ƭÑY 15ʁƎũŋMpZ˟sqX=pZZk]5¼ÆƝ_÷XBǃs\=ŔįkśE5
I_m>\ŔįʾƋ˦ǒ`ńY<o5ǼǴ˦ǒB˚Y<p6gR5 ɡ5
ȴˎƢɑˆ]mpɦɡ5ÛdzƢʙǐ5|·�µzy³�ƩdzɤʊZć
˦ǒBƟ˚Z\pIZB<o5ǼǴ˦ǒBǣĕY<p6 � ɡ]ôɠ_Ɂ=ŢƢBŤŦąǥɊ]¸Ǣ×ú]ɡzy³�]Ʃ
dzMpZ5P_āɓõ]òŞƢɡɤëʬCongenital Rubella Syndrome : CRS tɦɤMpIZB<o5ŝC\ľZ\p6CRS _ŝɤɋ`ɨú5
òŞƢƞɢƤ5ʱY<pB5P_Óʤʽɤ5ˎŹǯȭź5ʲʺʼ5˷ß@
mbʔɺ_ɦƐ\[tÜ>IZk<p6CRS _ǼǴ¹˦ǒ©�®w³`
8òŞƢɡɤëʬ9_]ˣM6 Ɖƭ 26 Ɗ 4 Ǣmo8Ku]MpɊůƩdzɤÊƺ9BɦĕJq5ɡ
_˦ǒ]@GpǼǴ˦ǒ_ƗďBmo˚Z\UR 1)6 ¡�� NMÚ�Öá�viU4�
�
¡� �� '�(Ñ'�t�
� ɡzy³�`�±�_ɣġß]ĂJqX=p_Y5P2 ŰǕˤY
BSL2 _ĩoư=Œȳ]ƙUXàǽtː>6 ˨ʯ`ÊjɡƳßåtȰůK5ƳßBɁ=AÞ=ŔįHI Ƴßå 16 ê
×¾]`ɡ¶��·tǁʂK5Ƴß_ɍƚl½Ǚtɷ˪KXAnǼǴ]
ƙËMpIZBǤgK= 2)6 ¡�¡�� NM�@Û�ÞA�
6
� ɡ_ǼǴ˦ǒZKX5ˎȫūɪǔȚ@mbɣġßǼāȚBĈɕīʵY<
p˓ 6ˎȫūɪǔȚZKX`5IgM ƳßǼāȚ5IgG ƳßȰůȚ@mb
˴ˎɑÿƲĉHIƳßȰůȚ\[BĈɕīʵY<pB5ơƢǥ_ˎȫt
ɕ=p IgM ƳßǼāȚB»˂ɪY<p6 � ɣġßǼāȚZKX`5ĻƸ=Ȩ5ˎȨ5Ż\[tǼß]KXzy³�
Ă@mbzy³�ÛŧǼāBīʵY<p6zy³�Ă`ĂİůgY
]ǜBAApIZ5@mbĂīʵ\ǼßǀĩǜǥBzy³�ÛŧǼā
mokɵǥY<pIZʊAn5gN`zy³�ÛŧǼāȚtː>IZB
»˂ɪY<p6 ¡�¢�� �eÛNMH?Ñ;�CF�
� zy³�Ă@mbzy³�ÛŧǼā_ǼǴǩǑZKX5ɡZù_
ʾƋǩǑĻƸ=Ȩ5ÿŇȉǧǺˎȨ5Ż3,4)BǂšJqp6ɡY`
ʻƸ=Ȩ5ȨkǼǴǩǑZKXâɕīʵY<p6ɡɊɞɪƳß_Ǽā
]`ˎȫgR`ˎȷtɕ=p6ǼǴǔȚ`ɣġßǼāȚZˎȫūɪ˦ǒ
Ț]ĂAqp6PqQq_ǼǴ]`ă\Ǽß5ǼßǀĩǜǥB<p�˓ ��6 ˓
ǼǴǔȚ Ǽß ǀĩ]ă\ǜǥ
cRT-PCR real-time PCR
ɣ
ġ
ß
Ǽ
ā
zy³�Ă
ĻƸ=Ȩ5ˎȨ5
Ż5ʻƸ=Ȩ5
Ȩ
ɦɤ2-3ǗċAnɦɤƘ1ʁƎ
IgM ƳßǼā
(IgM ELISAȚ)
ˎȫ ɦɤƘ4Ǘ;28Ǘɭ ˎ
ȫ
ū
ɪ
ǔ
Ț
IgG ƳßåȰ
ů (IgG ELISAȚʊ)5HIƳßȰů
ˎȫ¦wˎȫ 1Ńɭ:ɦɤċ;ɦɤƘ4ǗɭgY 2Ńɭ: 1Ńɭ_ǀˎAn2;4Ƙ5ɦɤƘ 2;4Ƙ
7
Iqn_Ǽß_ĀɒǔȚ_ãtƻHp6 1) Ļ@mb^F=ȨgR`ʻƸ=Ȩ zy³�Ăő@mbÛŧǼ
Ǵ įƭȅʶʚǩ_�¶¢ǀĩ��x��ãSterile Swab Applicators, Copan ɹ˗ YĻ<p=`ʻʓʽt^F=5zy³�çũ˼Ȩ
;3mL]ȦȶMp6zy³�çũ˼Ȩ`Ƅ˰ļãBD ¯��º�
³�±·�¨º�Ǽß˼ɕőŌ5BD ɹ˗_eA5ʿųˮǎȨBâ
ɕYCp6Ǽß` 3,000rpm, 20 ĂƞK5½ȫtǼǴ]ɕ=p6 0.5%z�ˎȫw³¢ª·¡±��°· VBSA 5¦��²· G
100-500U/mL 5��´£�©y�·100-500µg/mL 5�·�©y
�·100µg/mL@mbw·¡}�²�· B2µg/mLtȪđKR
ʜʴőɕőŌMEM őŌʊgR` PBS(-)tɕ=p6
2) ÿŇȉǧǺˎȨzy³�Ăő@mbÛŧǼǴ �|·��²z«gR` EDTA đǧǺˎȨtɕ=p2;5mL 6¤�
²·đǧǺˎȨ_Ŕį`5RT,PCR ħƠtŲMpRj5zy³�Ă
ő_h]äMp6ɡǼǴZİǜ]ː>Ŕį]`5ǀˎJqRÿŇȉ
ˎȨ`×¾_=NqA_Āɒljàtː=5ǧǺˎĝǷɑʜʴPBMCt
ĂKX˨]äMp6ɡǼǴ_h_Ŕį`5÷ˎ5ˎȫ5ˎȷtɕ=
Xk˃=6 ǔȚ ÿŇȉˎȨ 2mL ]ʊ_ PBS tȩĺMp6 <nALj Ficoll-paque ȑ 1.077 3mL tĂȜKRƞ�®º¢
] 2 êƆKRˎȨtA]žMp6 1,300)g520 ĂƞMp6 ˓žAn]ˎȷʘêƆ 5ǧǺˎĝǷɑʜʴPBMC 5ȑ
Ȩ@mb˴ˎɑƭĂ]ĂəJqp6PBMC `zy³�Ă@mb
ÛŧǼǴ]äMp6\@ˎȷ`zy³�ÛŧǼāĦbzy³�Ă
\nb]»_ƳßåȰů���tɕ=RƳßåȰů]âɕYCp6 ĂəJqR PBMC ` PBS YŃȟȢƘ5þʟçũȨ10%DMSO
8
đz�ʳõˎȫFBS ]Ʈů_ȻƎ2)104;2)106 cells/mlYˮǎK5-80+YþʟçũMp6İǿ]ˎȷ`-20+»`zy³
�ĂɕZKX-80+]çũMp6
ǔȚ ÿŇȉˎȨt 3,000rpm520 ĂƞMp6 ƞJqRȨ_½ž`ˎȷəĂ5¾ž`ˎɑəĂ_ž]ĂəJqp6
ˎɑəĂ˓ž_ɨ˄tĶMpʜʴƭĂt�³µº�x·���£ʊ
]moŃĨMp6 ŃĨJqRʜʴtñ__zy³�˼çũȨgR`ʜʴʣƹɕő
Ȩ]ûȤKXǼßZMp6 P_Ó_əĂ`ǔȚ]ȳLXçũMp6
ǔȚ ˎȷƭĂZ²·�ɑ_ĂȚ
� ˎȨtűȯY ����&%"5�� ĂƞMp6�
� ˎȨƭĂBȩöK\=m>½ȫˎȷƭĂtŃĨMp6�
� ˎȨƭĂ]ǀˎ_ �êZ\pm>] ��� tđ?pǀˎB "!
\na5��"! Z\pm>] ��� tđ?p 6�
� �y·�µº�ºtɕ=XűȯY ����&%"����������5�� Ăƞ
Mp�
� ½ȫ�êƆJqRˎȷƭĂZ²·�ɑĂȨ_Ŗɚ]ƖƭJ
qR²·�ɑžtŃĨMp6�
� ŃĨKR²·�ɑ]ê×½_ ��� tđ?5űȯY �������5��
ĂƞKXȟȢMp6I_ljàtˡŃː>6ˎȷƭĂ5²·�ɑ
tçũMp6�
3) Żzy³�Ăő@mbÛŧǼǴ
10 An 50mL _ŻtǀĩMp61,000;1,500rpm510 ĂƞKRȗȮ
ʜʴt 2;3mL _zy³�˼çũȨ]ûȤJORk_tɕ=p6ȗȮ
ʜʴBƚnq\AURŔį]`ŻǼßtP_ggâɕMp6zy³�Ă
]ǦĀɒŻǼßtâɕMpŔį5P_ggSZʜʴŲB˵CpIZB<
pRj 2~10 ê]ƆKXâɕMp6
9
4) ʸʷȨzy³�Ăő@mbÛŧǼǴ5ƳßåȰů ǀĩJq
RǼßtP_ggɕ=p6 � ˎȫ5ˎȷƳßåȰů ˎȨǀĩƘ5ĂKXâɕMp6�
�
�� ˎȫ5ˎȷzy³�ÛŧǼǴ�ǼßAn ����"%���&�!�����"�#�� �'�
��w�·ɹ��ʊY ����tƵāKâɕMp6�
7) ÷ˎ (zy³�ÛŧǼǴ): Nuclospin RNA Blood (��±�y~ɹ)ʊt
ɕ=X ÷ˎmo RNA tƵāKâɕMp6 ¡�£�� NMH?Û��Øn�
ǼßtǀĩKRn+]çʍK5lA]ǼǴŰǕǕˤcýˈɋƪY˼M
p6ǀĩƘ 48 ǜ×ú]zy³�ÛŧǼāȚ<p=`zy³�ĂȚB
ťYCpŔį]`P_gg 4+YçũMp6ǼǴťBǀĩƘ 48 ǜt˶?
pŔįlǥçũ_Ŕį]`-80+]çũMp6ˎ ȫūɪǼǴ]ɕ=pˎȫ`
���+YçũMp6 ¡�¤�� NMÛ�&�
� Ȉ_=NqA<p=`˘Ǎ_ʟDzBƚnqRŔį5ɡzy³�ƩdzƢ
ZĆůMp6 ɡzy³�BĂ¹İůJqp ɡzy³�ÛŧBǼāJqp6 ɡzy³�Ɋɞɪ IgM BǼāJqp6 ¦wˎȫơƢǥ@mbŃƛǥ]@=Xɡzy³�Ɋɞɪ HI Ƴß
<p=` IgG Ƴß _˸5gR` HI Ƴßå_ê×½_½Ǚ 2) <
p=` IgG Ƴßå_ê×½_½ǙB˪jnqp6
� IgM Ƴß_Ȱů`Ƈ]ǣɕY<pB5ƓƢ_Ŕį`ƟNKkǡ˿_
ɡzy³�ƩdztħǛMp˥Y`\=_YȜƧBƟ˚Y<p6ƢƩdz_Ŕ
į5ɦɡBāɐKXAnEZkǗƘ]` HI Ƴß@mb IgM Ƴß_½Ǚ
B˛nqp6]5ɦɡɮƘ0;3 Ǘ_ˎȫY`Ŕį]mUXƢZYp
10
IZB<p_Y5ˎȫ_ǀĩǜǥ]`ȜƧt˚Mp6 ¡�¥�� 5LgT*Õ� ZÚÔÕáNM�@Û�à7Ó�
� ɡ`ƩdzɤȚ]@GpÍƩdzɤ_>T÷ǍƱdžƩdzɤ]ƺůJqX@
o5÷X_Ěɥȇ`ɡƤʯt˦ǒKRŔį5żātː>IZBůjnq
X=p6ɡ_żāŒȳ]@=X`58÷˷Ƣ_Źʙǐlʙ˄Âɡ958ɦɄ9
@mb8²·�ʏʼʹ9_VMdX_ʾƋɤɋtɸKRɤãtʾƋ˦ǒã
ZMpZůjnqX=p6KAK5¼ƝëBMdXPrs\=ŔįkśE5
P_Ŕį`żāŒȳtȲRMRj]ǼǴ˦ǒBƟ˚Z\p6 ¢�� zX$iNM�
¢� �� �z`��8�ą±¾Ã»¿¿ÂËÊÁÄ»ÊÁÅÄ�²ÄÀÁ¼ÁÊ ÁÅÄ©�±²Ć9�Y&T ¥�§��
����������
¾ˣ]ˮ˗ǔȚtɸM6=NqkƄ˰ļB<qaÕǠYCp6 �~²·Āɒɕ˨ˉ
1. PBS(,)Dulbecco _ PBSpH7.2;7.4 NaCl 8.0g, KCl 0.2g, Na2HPO4 1.15g, KH2PO4 0.2g tˇɛȓ]ȴ
˞K÷t 1,000ml ZMp6 2. 25%�~²·ƬȺȨ
�~²·Fisher gR` Aldrich ˗25g ] PBS(,) 100ml tđ?
Xà˗Mp6�~²·` PBS(,)Y 3 Ńȟ>6½ȫ_ pH B 7 ×½
Y<pIZtɷ˪KRn5Ȩ_JtLjƷċ]VGRĞ]<sO
p6 ˴ˎɑɕ˨ˉ
1. DGVDextrose-Gelatin-Veronal NaCl 8.0g5¥µ�º³ 0.575g5¥µ�º³-Na 0.375g5CaCl2 0.02g5MgSO4¹7H2O5�±�· 0.6g5¢�zʕ 10g tˇɛȓ]ȴ˞K5
÷t 1,000ml ZMp6 2. ��°z˴ˎɑȤȨ
��°z˴ˎɑ`Ƅ˰JqX=p6Pqt DGV YŃȟUR<Z5
DGV YȤȨtà˗K5 +]çũMp6ǀˎƘ 3;4 ç
ũYCp650ȤȨ`âɕɮċ]58ȤȨt 1500rpm 10 Ă
11
ƞK½ȫtƿXRƘ5ʊ_ DGV tđ?à˗Mp6HI ˨
Y`Ƙ_ƆȨY 0.25ȤȨtàoâɕMp6
ĮʂƆȨ 1. PBS pH.8
NaCl 9.0g5Na2HPO4 0.67g5KH2PO4 0.72g5NaN3 1.0g tˇɛȓ
]ȴ˞K÷t 1,000ml ZMp6 � � � 2. �³�z«©���z«Ȩ
CaCl2 4.0g5MgCl2¹6H2O 4.0g tˇɛȓ]ȴ˞K 100ml ZMp 3. 1�±�·
�±�· 1.0g ]ˇɛȓ 100ml tđ? 115+10 ĂŊȵˆMp6Ź
ĂĂȜK54+]Xçũ6âɕǜ]ȯȥYZAM6 3. ƆȨƳġ@mbˎȫƆȨ
PBS pH6.8 200ml ]Ɉˎȫw³¢ª· 0.2g51�±�· 1.0mltđ?Rk_tâɕMp6
4. ƆȨ˴ˎɑȤȨà˗ɕƆȨ ƆȨ 200ml ]_�³�z«©���z«Ȩml tđ?p6
HA˴ˎɑÿʚHemagglutininƳġ HA Ƴġ`þʟÈɅJqRk_BƄ˰JqX=p�·�ɓɶʊ 6
¢� �¡�� zX�a�
� ˎȫÅ]` HA ħƠtƲĉMpɊɞɪy·���ºBũŋMp_Y5Ê
j�~²·Āɒ]mUXIqtĩoE6gR˴ˎɑ]ŷMpʿɂÿʚ`5
ŝ_˴ˎɑYĵĨMp6ïĘJqRˎȫY`5y·���ºBG\=
Ŕįk<p_Y5ïĘKRˎȫ`ɕ=\=6ƳßƢZƢ_Ĥʮˎȫk
İǜ]ĀɒMp6ˎȫĀɒ`×¾]ɸM©y�µ£´º�gR`©y�µ�
®º¢Yː>6 ćĆùäèā÷ĀĄòT�
i) Ɖƌ©y�µ£´º�_ʃt»V@C]â>6˔ǼˎȫĮ 25*l töqp
ņ 6 ii) 25%�~²· 0.1ml NVtˎȫ_öUX=pʃ]=qp6�~²·B¿Ŏ
»]\n\=m>]5ǜ7�~²·ȨtûȤJOʚǘEĂȜMp6
12
iii) £´º�½_ȓĂtƸCĩUXAn�º³Mp6 iv) £´º�tƯYȹKEƼp6űȯ] 20 ĂNjʪMp6 v) £´º�tƼURƘ5£´º�ƞȇY 1500rpm53 ĂƞMp6 vi) ƞƘ`£´º�tȓƉ]çƹK�º³ʃîtȼnJ\=m>]K5�
º³t`BM6 vii) Įʃ] 8%˴ˎɑȤȨt 50*l @mb PBS(-)t 25*l đ?p viii) ûb�º³tK5£´º�t˹EƼUX˴ˎɑtmEȩĺJOp6 ix) £´º�t 4+] 1 ǜAn 1 Ŝ@E6I_ 2 Ǝ£´º�tƼUX˴ˎ
ɑtûȤJOp6 x) £´º�tƞ1500rpm53 ĂKXAn�º³t`BM6I_½ȫ
BĀɒȬh 1:8 ƆˎȫZ\p6
ņ� ©y�µ£´º�Y_ˎȫ_�~²·Āɒ Ɖƌ©y�µ£´º�_ʃt 1 V@C]â>6
�
ĈĆùäèāðûĄöT�
i) ˎȫ 50*l t 1.5ml _©y�µ�®º¢]Zp ii) PBS-t 150*l đ?p iii) 25%�~²·Ȩt 200*l đ?5ȹKEƼp6űȯ 20 ĂNjʪ5P_] 253 ŃɬMp iv) 2000rpm20 ĂƞƘ5½ȫtć_©y�µ�®º¢]ɿM6 v) 50%˴ˎɑȤȨt 12.5*l đ?5Ȕȥ] 1 ǜ@Eĥ` 4+1 ŜNjʪMp6
13
I_5ǜ7�®º¢tƼUXˎɑtûȤJOp6 vi) 2000rpm520 Ă 4+YƞƘ5½ȫtŃĨMp6Iqt 1:8 ƆˎȫZM
p6 ¢� �¢��±¬ 9�Û&��
HI˨tŰǕMpċ]5âɕMpHAƳġ_ĐåtȰůK\EX`\n\=6 1) ƆȨ 1 tɕ=X HA ġȨt 1:2 ]ƆK5P_ 50*l t 96 ʃ U ƌ©y
�µ£´º�_ʃ No.1Ȅ]@=RŔį_ǡkƂʇ_Ą]Zp6İ
Lk_tĄVEo5HA Ɔt 2 Ąİǜ]ː>m>]Mp6gRƟ˚ m
o˩ʜ] HA ůtŰǕKR=Ŕį]]ƠLXġȨt 1:3 @mb 1:10]ƆKRk_kĄİǜ]ƆMp6
2) ƆȨ 1 t No.2 An 12 gY 25*l NVđ?p 3) 2 êȍƆt No.11 _ĄgYː>No.12 _Ą`ˎɑŷɃZKXâɕ 4) ƆȨ 1 tĮʃ] 25*l NVđ?p 5) Įʃ] 0.25%˴ˎɑȤȨt 50*l đ?p 6) £´º�tMF]©y�µ£´º�ª��º\[tɕ=5mEȩĺJOp
ɽNVŃ 6ˎɑ_ȩĺBěĂY\=Z¿ŭ÷ÿBʢC HA _ů
BȊɷ]ː?\E\pŔįB<p6 7) £´º�týˈƏ4-10+]ǜʪE 8) £´º�tűȯ]SKX 5-10 ĂƘ]£´º�t˝ŶKʟDztĆůMp6
ŭ÷gR`ĂÿKRʝǧ_ƆêǍt5P_HAġȨ_HAåZMp6
I_ìt T ZMpZ5HA ġȨt T/4 ê]ƆȨ 1 YƆKRȨ4 ĝÝ
HA _Đå]ɯƕt HI ˨]âɕMpƳġZMp6ã?aġȨ_ HAåB 32 _Ŕį]`ġȨt 8 êƆKRk_t HI ˨]âɕMp6
¢� �£��±² G���
U ƌ©y�µ£´º�_ʃtņ_m>]ƼoĂGp6
14
�
ņ� Ǽß@mbŷɃ_©y�µ£´º�½Y_´ywz�ã�
�
1) ĀɒȬhˎȫt<HI ˨>_ 1:8 _ʃ] 50*l5<ˎȫŷɃ>_ 1:8 _ʃ]
25*l Zp6Ó_ʃ]`ƆȨt 25*l NVöqp6 2) 2 êȍƆtː> 3) <HI ˨>_ʃ]ˮ˗KR HA Ƴġ 25*l NVt5<ˎȫŷɃ>_ʃ]Ɔ
Ȩt 25*l NVđ?p6©y�µª��ºʊYȩĺƘ5űȯ] 1 ǜNj
ʪMp6 4) HA Ƴġ_ 2 Ȉůtː>6<HA ÌȈů>_ 1:1 Ɔ_ʃ]<HI ˨>
_ʃ]đ?R_ZİLƳġt 50*l öqp6Ƙ`ò]dR HA ˨_˚
YêȍƆŰǕƘ5ƆȨt 25*l đ?p6 5) 0.25%˴ˎɑȤȨ 50*l NVtMdX_ʃ]đ?ěĂ]ȩĺMp6 6) £´º�týˈƏ4-10+]ǜʪE6 7) <HA ÌȈů>Y HA åBĝÝY<pIZtɷAjp6ƳßƢ@mb
ƢŷɃtˤůKX=pŔį`5ŷɃˎȫ_ HI åBȊKEYX=pAt
ɷ˪Mp6×½BȲRJqRŔį5˨ƭʅZMp6ˎȫŷɃ]@=
XʿɂÿʚBũŋMpǼß`5ˎȫĀɒtûƎŰǕK5ûǼǴMp6 8) HA ħƠtŭ÷ȉKRǡʝƆêǍt5P_ˎȫ_ HI ƳßåZKXĆ
ůMp6
15
¢� �¤�� �viÙU4�
HI ƳßȰů]Ɵ˚\˨ˉB���]\UX=pǼǴ˨ˉk˰ŘJqX@o
âɕBīʵY<p �·�ɓɶ ɡzy³� HI ˨ 8ɓɶ95R-HI8ɓɶ96
I_m>\ǼǴ���tâɕMpŔį]`���_âɕˬǚǟ]ƙ=ljàĆ
ůtː>Ɵ˚B<p6 �
¢� �¥�� �Ûj%�
HI ˨Y`5ɡɊɞɪ IgM, IgG @mb IgA _=NqkħƠK5ʦįɪ\ ƳßåZKXȰůJqpIZAn5ʟDz_˞]`ěĂ\ɒ˞BƟ˚Y<p6 IgG BȰůJqpIZAn5Ʃdz_˦ǒɭɪ_âɕ]`¦wˎȫtɕ=pƟ
˚B<p6ơƢǥ@mbŃƛǥ_ 2 ɀ_ˎȫtȰůK5˸<p=`ê×
½_Ƴßå_½ǙB˪jnqqa5ɡzy³�_ƩdzB<URZĆǒYC
p6»ǔY5HI ˨Y` IgM kǼāMpIZAn5= HI Ƴßå`ɮ
˿_ɡƩdztħǛK>pīʵƢB<o5P_Ćů]`ʾƋɤɋʊtĴjě
Ă\ǼˢBƟ˚Y<p6ƢƩdz_Ŕį5»˂]ɦɡBāɐKXAn 4 ǗƘ
]` HI Ƴß_½ǙB˛nq5ɦɡāɐƘ 1-2 Yǡå 1:256 An 1:8192ʁƎ]Mp67 ¸ǢAn 3 ƊƘEn=gY`5128 êʁƎ_ƳßåYŬ
ůMp6512 ê×½_ƳßåBɦɡāɐƘ 7 ¸Ǣ×½ƹʢMpIZ`\=Z
ʮ?nqX=p6ƙUX 512 ê×½_Ƴßå_Ŕį`5ąƩdzƘ 7 ¸Ǣ×ú
A5ûƩdz]mp¢º��ºY<pīʵƢB=6 ¢�¡��²¿³ �p d9�Y&� �²¿³�®²¬��
ǁȚ\nb] IgM ƳßƾƽȚ]mp EIA ���Z_ʂB<oƄ˰J
qX=p6=Nq_���kâɕˬǚǟ_ˣ˻]ƙUXljàKĆůMp6 ¢�¡� �� �Ûj%�
���_ĆůŒȳ]ŒWCĆůtː>6ąƩdz_Ŕį5ɡzy³�Ɋɞɪ
IgM `ɦɡāɐƘǍǗYǼāJqťj5ɦɤƘʁƎY º�]K5
ɦɤ¸ǢƘgY]ǼāJq\E\pZJqp6ɦɡāɐǗgY`5ěĂ
] IgM BɔɓJqN5ʘĜǍYǼāJq\=IZ]ȜƧBƟ˚Y<p6ƹʢ
ǥ]V=X`éÑƃBŝCE5ǥ]sRUXƹʢƢtɸM�º�k<
pƓƢ_IZBś= 6gR5Ó_Ʃdzɤ]mUXɊɞɪ]ƓƢZ\
pIZk<p6
16
ûƩdz_ŔįYkɤã_ʘ 50] IgM ƳßBǼāJqp_Y5IgM Ƴß_
ǣɁSGY`ąƩdzZ_Ćć`ńY<p6»˂]ûƩdzãY`5ƹʢǥ
BɵEƳßåkÞ=6 ¢�¢��²¿° �p d9�Y&� �²¿°�®²¬��
ǁȚ_ EIA ���BƄ˰JqX=p6=Nq_���kâɕˬǚǟ_ˣ˻
]ƙ=ljàKĆůMp6 ¢�¢� �� �Ûj%�
ơƢǥ@mbŃƛǥ_¦wˎȫ_Ƴßåtİǜ]ȰůK5Ƴß_˸<p=
`5ê×½_½ǙB<URŔį5ɡzy³�ƩdzB<URZĆůYC
p6
17
£�� ��#$iNM�
ǨʆY`ǼßkKE`Ăzy³�An_ɡzy³�ÛŧǼā@mb
Ûŧŏ˞ǰȚtˣ˻Mp6 ɡzy³�Ûŧ_ǼǴ]`5TaqMan £µº¢tɕ=R²w³�y«
RT-PCR Ț5gR`�·¥·�°�³ RT-nested PCR Ț Ǿˋɨ˳ NSÛŧŗƈȚBĈɕYCp6²w³�y« RT-PCR Ț`ʒæƢ5˾Ƣ]
MFq5ÀVŰű�·�ª�º�°·_²��BÞȭYCpIZAn5ʉ
»ƴZKXǂšJqp6»ǔY5²w³�y« RT-PCR Ț`�·¥·�°
�³ RT-nested PCR ȚNS ÛŧŗƈȚZȑ˺KXllƩƎBĒpIZ
An5moĢŵ\ĆůBȕjnqpŔįòŞƢɡɤëʬ_¡}µºw�
£ǼǴ5ŤŦŢƢ_ɡʫƤ_˦ǒʊ]`5�·¥·�°�³ RT-nested PCR ȚtƴMpIZBǤgK=6 Jn]ɡzy³�ƢY<URǼß]V=X`5�·¥·�°�³
RT-nested PCR ȚE1 ÛŧŗƈȚYŗƈKRɔɉtɕ=X5Ûŧ
Ą˞ǰ@mbÛŧŏȖůtː>IZBǂšJqp 1)ņ 6
ņ� ɡzy³� RT-PCR ŗƈÝ
18
£� ��¹»Ç³»Ä ÷āĄöâcÓ×þãÿîäú ·¹�¶· T�
˸üħƠ@mb PCR ħƠt�·�³�®º¢Yː> TaqMan £µº¢t
ɕ=R one-step ²w³�y« RT-PCR Țtˣ˻Mp6IIY`5ãZKXw
£±y��y~���«�ɹ_TaqMan Fast Virus 1-Step Master Mix@m
b Applied Biosystems 7500 Fast ²w³�y« PCR ���«tɕ=RǔȚ
tɸM6Ó_˨ˉ@mbȇłtɕ=pŔį]@=Xk5×¾_Ư]ȳLX
ŰǕMp6Ǩ Real-time RT-PCR Ț`5ɡzy³� Real-time RT-PCR Ț
4)ZİLħƠǫÙȯƎ@mbħƠǜY<pIZAn5İ»_£´º�Y
ÄːKXŰǕMpIZBīʵY<p6 £� � �� �Ù�wăQ��
zy³�RNAƵā���ã: QIAamp Viral RNA mini kit ; �w�·ɹ
Real-time RT-PCRɕ©��ºª���ã: TaqMan Fast Virus 1-step Master Mix ; w£±y��y~���«�ɹ
©y�µ ¦��ĮŴ_k_
¡x³�ºÔC ¦����£½ˣ_©y�µ ¦��]ŷƠKRk_
1.5ml |�¦·�³¡�®º¢ǷÞĵɳ�y£ã ������$��#���(���
��"�|�¦·�³¡ɹtǂš
1.5ml |�¦·�³¡�®º¢ɕýğƞȇ 96well reaction plate (ã: Fast 96-well Reaction Plate (0.1mL); w£±y�
�y~���«�ɹ) £´º��º³ (ã: Optical Adhesive Covers; w£±y��y~���«
�ɹ) Ûŧŗƈ˕ʪ ã: Applied Biosystems 7500 Fast ²w³�y« PCR ��
�«; w£±y��y~���«�ɹ RNase free ȓ £±y©º@mbǼāɕ£µº¢ £±y©ºȜ
NS(32-54)Fwd: 5’- CCTAHYCCCATGGAGAAACTCCT -3’ (10µM) NS(143-160)Rev: 5’- AACATCGCGCACTTCCCA -3’ (10µM)
19
Ȝã±y©º_<nALjª���Į 10µMKRk_tˮǎK
Xk˃=6P_Ŕį-20+çũK5þʟˍ˞tǍŃ×ú]Ʋ?XâɕMp6 ȩįŕŒ H ` A, C, TG ׌, Y ` C, Tpyrimidine ŕŒZMp6
£µº¢Ȝ NS (93-106) Probe: 5’ FAM-CCGTCGGCAGTTGG-MGB 3’ (10µM) Ȝ10 µM ]ˮ˗KRk_t RNase-free _ȵˆ�®º¢ʊ]ĂȜK5
þʟˍ˞tǍŃ×ú]Ʋ?XâɕMp6
Ƣ�·�µº³ ¹ɡĤɃ RNA Ȝ
ȜňʅƩdzɤɶʄƮzy³�ʉ¼ʉÌűmoƅMp6 £� �¡� �e�\·´¬âcÓ×��ÛD���
� Ûŧŗƈ˕ʪ_ȇʂlµ��]mUXǼāƩƎBsNA]ɞ\pIZB
<p_Y5ąjXŰǕMp]`×¾_ª£� �¡� «�ª£� �¡�£«tĮǕˤ]
@=XƟNː=5˨ʖtǡĘMp6gR5ůǥɪ]gR`Ɵ˚]ƠL
XŰǕMpIZ]mo5ȇł_ƩƎ�{��]\p6 £� �¡� � �e�\ ·´¬ Û,� . ɡĤɃ RNA _ġȨ1×106 � º/5µltȔ½Yȴ˞KRƘ5LjƷK
spin down Mp6 / �®º¢ 1 Ǩ] 95 µl _ȓ<p=` RNA Ɔɕ��¡vºã��±
�y~ EASY Dilution for Real Time PCRtĂȜK585×1049Z±¥³M
p6 0 �®º¢ 4 Ǩ] 90 µl _ȓ<p=` RNA Ɔɕ��¡vºã��±
�y~ EASY Dilution for Real Time PCRtĂȜK585×103985×1029
85×101985×1009ZPqQq±¥³Mp6 1 ɡĤɃ RNA _ġȨ1×106� º/5µl 5µl t585×1049�®º¢]đ
?5LjƷK spin down Mp5×104� º/5µl Z\p 6 2 85×1049�®º¢_ 10 µl t85×1039�®º¢]đ?5LjƷK spin downMp6 3 2Zİǿ]585×1039An85×1029585×1029An85×1019ZɿKX=
20
C585×1009gY_ 10 êȍƆtà˗Mp6 4 85×1049 20 µlNV]ĂȜƘ5-70+×¾YçũK58ɡĤɃRNA 5×104
� º/5µl 9ZKX5ȈŃ×_ǡĘ]ɕ=p6P_`5½ˣ_2×
_Ɔtː>6 £� �¡�¡� �2VÛ�{�
×¾5Applied Biosystems 7500 Fast ²w³�y« PCR ���«tâɕ
_Ŕį . £±y©ºª���5£µº¢54×TaqMan Fast Virus 1-Step Master Mix tȔ½Yȴ˞K5ȩĺKX@E6 / Ȕ½]�®º¢tʪC5×¾]ƙUX57 z{³Ăz{³ĂĎ
_ħƠȨtˮ˗Mp6
�
�2Ò
×à�ÏÂ��
¦ �2�~
�ÏÂ�� r[0�
·´»É¾�¯È¾¾�ͻʾÈ� ¤�¨� £ �¢� �
£Ð¹»Ç³»Ä�¯»ÉÊ�ºÁÈËÉ� �ÉʾÆ�³»ÉʾÈ�
³ÁÎ� ¤� ¢¤� Ð�
´¸�¢¡�¤£�¯Í½�� �ϳ�� �§� ¡�¥� ¨��ij�÷ýäùĄ�
´¸� £¢� ¥��·¾Ì�� �ϳ�� �§� ¡�¥� ¨��ij�
� �
�
�
�
� ´¸��¨¢� �¥��¶Èż¾�� �ϳ�� ��¤� ¢�¤� ¡¤�ij�
� �§ åæÿ�Ú�U� ¤� � �
ëĂ÷ÿ ·´¬� ¤� � ��
� � �~� ¡�� � �
0 96well reaction plate _ 6 z{³]5/_©��ºª���t 15 µl NV
ĂȜMp6 1 RNase-free ȓ@mb8£� �¡� �e�\ ·´¬ Û,�9Yà˗KRɡ
ĤɃ RNA 85×104985×103985×102985×101985×1009_ 5 ʂt 5 µlNVŷƠMpz{³]đ?p6 5×104;5×100� º/ħƠZ\p 2 £´º��º³YŸtKRƘ5ħƠȨt˹E� ·�z·Mp6
21
£� �¡�¢� þãÿîäú ·¹�¶· �2�
Applied Biosystems 7500 Fast ²w³�y« PCR ���«]���K5×
¾_ǫÙYħƠtː>6 / %�/
Assay/ Absolute Quantification (Standard Curve)/Run Mode/ Standard 7500/Reporter/ FAM/Quencher None Ȝ
�� �� KGJT�
1. ('��� 509 5� 1
2. ('�+ �� 959 20� 1
959 15� 3. PCR��
609 1� 50
Ȝ 5ɡzy³���«Ǽāɕ£µº¢`ˌó�|·�ºtȂ˯KX
=pRj5�|·�ºZKX TAMRA tȂ˯KRɡzy³� N ÛŧǼ
āɕ£µº¢ 4)Z`ˤůBɞ\p6Reporter @mb Quencher `z{³ȏ]
ˤůYCp6 £� �¡�£� ñĄî|J�
ɌʅKR3Ń_˨tŰǕK5×¾_V_ŒȳtȲRKX=pIZtɷ˪M
p6 . ȍƆKRɡĤɃRNAYà˗KRǼʧ_slopeB-3.1An-3.8_
Y<pIZ6 1�y�³Y2ê_DNAǒɆ]ŗƈMpɒƦɪ\PCR ħƠ_Ŕ
į5slope`-3.32]\p6 / ȍƆKRɡĤɃ RNA Yà˗KRǼʧ_ R2ìB 0.98 ×½Y<p
IZ6 0 5×101� º/ħƠ_ɡĤɃ RNA B Ct≦40 YǼāJqpIZ6 1 Ƣ�·�µº³Y<pȓBƢCt>40Y<pIZ6 RSK5Ǽʧ` Ct=40 ×úYǼāYCRƆgYYà˗Mp Ƈ55×101
;5×100� º/ħƠ 6 Iqn_ǫÙtŬůKXȲRJ\=Ŕį`5ȇł_ǵȊ5˨ˉ_ûˮǎ\
22
[tǼˢMp6 £� �¢� NMÛ'B�
£� �¢� � åäÿí·´¬:�� �
zy³�RNAvRNA_Ƶā`���ȪÔǏǟ]ȳLXː>6 İǜ]ȓtǼßZKXRNAƵātː=5RNAƵā_Ƣ�·�µº³ZKX
âɕMp6 ƵāRNA`5-70+×¾YçũMp6 £� �¢�¡� �e�\·´¬Û�=�
5×101 @mb 5×100 � º/ħƠ_ĤɃRNAƆȨÌɀtƢ�·�µº³
ZKXâɕMp6ĮǕˤ_ɋș]ƠLXȻƎtřǞYCpB5ȻƎĤɃ
RNA`5×101� º/ħƠt˶?\=m>]Mp6�·�ªtGpRj5
ȻƎ_ɡĤɃRNAtƆMpàǽ`5˨Zć_ŔƮYː=5ĂȜKX
-70+×¾YçũMp6 £� �¢�¢� �eåäÿíéõúÛN��
¾˓]ŒW=XƟ˚\˨ˉtˡʌMp6Ǽß׌]Ɵ˚\�·£³`5.
Ƣ�·�µº³ȓ 5/RNA Ƶā_Ƣ�·�µº³ȓtǼßZKX
RNA ƵāKRk_ 50Ƣ�·�µº³ÌɀġĊZKX 5×101 @mb 5×100 � º/ħƠ_ɡĤɃ RNA_ 4 VY<p6ħƠȨ_ˮǎ@mbħ
ƠǫÙ`ĉ£� �¡�¢� þãÿîäú ·¹�¶· �2ĊZİǿ]ː>B5Ćů]
` Ct=40 gYKAâɕK\=_Y5PCR ħƠ�y�³Ǎ` 40;50 _ʐŅY
řǞBīʵZMp6 � �2Ò×à�ÏÂ��
·´»É¾�¯È¾¾�ͻʾÈ� ¤�¨�
£Ð¹»Ç³»Ä�¯»ÉÊ�ºÁÈËÉ� �ÉʾÆ�³»ÉʾÈ�³ÁÎ� ¤�
´¸�¢¡�¤£�¯Í½�� �ϳ�� �§�÷ýäùĄ�
´¸� £¢� ¥��·¾Ì�� �ϳ�� �§�
´¸��¨¢� �¥��¶Èż¾�� �ϳ�� ��¤�
ëĂ÷ÿ ·´¬� ¤�
�~� ¡��
23
�� �� KGJT�
1. ('��� 509 5� 1
2. ('�+ �� 959 20� 1
959 15� 3. PCR��
609 1� 40~50
£� �¢�£� ��6kI��
Ƣ�·�µº³ȓ5ȓtǼßZKX RNA ƵāKRk_BƢħƠ
ɁK<p=` Ct ì>40Y<o5Ƣ�·�µº³5×101 � º/ħƠ
B Ct-40 Z\URŔį]˨BƭʅMp6 £� �¢�¤� �&�
Ƣ�·�µº³ZKXɕ=R 5×101 @mb 5×100 � º/ħƠ_ĤɃ
RNA ƆȨÌɀ_>T55×100 � º/ħƠB Ct-40 _Ŕį` 5×100 �
º/ħƠt55×100 � º/ħƠB Ct>40 _Ŕį` 5×101 � º/ħƠtĆů
ɕƢ�·�µº³ZKXâɕMp6 Ǽß_ Ct ìBĆůɕƢ�·�µº³_ Ct ì×¾_Ŕį]8Ƣ95Ćůɕ
Ƣ�·�µº³_ Ct ìmoŝCEÀV Ct ìB 40 ×¾_Ŕį]8Ćůç
ɛ95Ct ìB 40 moŝC=Ŕį]8Ƣ9ZĆǒMpņ 6 8Ƣ9ZĆůJqRŔį54-3� ɡzy³� E1 Ûŧ_Ǽā@mb
Ûŧ˞ǰ]ȳLXɡzy³�_Ûŧ˞ǰtŰǕMpIZBǂšJqp
6)68Ćůçɛ9_Ŕį]`5Ɵ˚]ƠLX5Ó_ă\Ǽßtɕ=XûǼǴ
tː>A5gR`İ»_ǼßY 4-2 �·¥·�°�³ RT-nested PCRNSÛŧŗƈȚ 55.zy³�Ă53.ˎȫūɪǼǴ\[tɕ=XĆůtː>Z
˃=6
24
ņ� ²w³�y« RT-PCR _Ćů¡µº�º�
£� �¢�¥� òýöÿÝÛ)2�
˨tːURʟDz5Ƣ�·�µº³BǼāYC\=ʊ_ľB˪jnqR
Ŕį`5ĂȜKRć_�·�µº³tâɕMpʊ5ă]ŷƠMp6˨ʖ
]ľB˪jnqRŔį`430102/-�� 876 F�<@&.B�)5tĤ
ɃKX˨ʖ_û˧åtː>6
25
£�¡�� êĂøĂìüóÿ ·¹�ľÉʾ½� ¶· Tċ�P�xh�ą´¸Ć��#"
-T�
Ǩ©�®w³Y`5ãZKX RT ħƠZ 1st PCR tĂGXː>ǔȚ Two-stepȚZʢKXː>ǔȚOne-step Țtˣ˻Mp6Two-Step ȚY`5
ɡ˦ǒ©�®w³ʉ 3 ɇ458zy³�ÛŧǼāȚ9]ˣ˻_ǔȚZù
_˨ˉtâɕMpǔȚtˣ˻KR6Ó_˨ˉtâɕMpŔį5ɡzy³
���«` GC ĴBƇ]=ʘ 70%Rj5 GC Ĵŏŗƈ]ŷ
ƠKR˸üħƠ˨ˉ@mb PCR ˨ˉTakara Bio ɹ Tks Gflex DNA polymerase ʊ_âɕtʮƫ]öqpZ˃=6 �
£�¡� ��¹ÍÅ�ÉÊ¾Æ TÚßá�eåäÿí ´¸ ��#ÛN��
£�¡� � ��w�
RNA Ƶā���»˂ɪ\ RNA Ƶā���@mbzy³� RNA Ƶā
��� ã High Pure Viral RNA kit µ�®w£±y��y|·�ɹ
tɕ=pIZBāǬp6 ˸üʚ [ãPrimeScript RT reagent kit (PerfectReal time)��
±�y~ɹ] PCR ʚ [ã PerfectShot Ex-taq (Loading dye mix) ��±�y~ɹ] NS Ûŧŗƈɕ£±y©º ɕ £±y©º
ı
˪ ˯ Ő
bp
ǔIJ Ą
NSL F3 5746-5762 F TCC TTG CGC CGA AGA CT 1st PCR
NSL B3-6 5984-6001 R AGA GGG GGT CCA CTT GAG
F2 nest 5811-5829 F CCA CTG AGA CCG GCT GCG A Nested
PCR B2 nest 5948-5967 R GCC TCG GGG AGG AAG ATG AC
FForward primer5RReverse primer
w�µº��³1.5~2.0% DNA Ăŧ©º�ºã100bp DNA Ladder �
£�¡� �¡�� åäÿí ·´¬ :��
RNA Ƶā`²w³�y« RT-PCR ȚZİǿ]ː> £� �¢� � åäÿí ·´¬
26
:�]ȳLp 6 £�¡� �¢�� ����2ą·¹Ć�
� IIY`5ħƠ]Ɵ˚\˨ˉB���_Å]MdXĴgqp PrimeScript RT reagent kit (Perfect Real Time)��±�y~ɹ˗tɕ=RǔȚtˣ
˻Mp6 ċ4-2-1-2YƵāKR RNA ȴȨ 10µL, 5)PrimeScript Buffer 4µL, PrimeScript RT Enzyme Mix I 1µL, Random 6mers 4µL, RNase Free dH2O 1µL tȪđKXʦ 20µL ]KRƘ537+15 Ă585+5 ɽ_y·�®
¥º�°·]mo cDNA įƭZ˸üʚ_¿ȠĘtː>6 RT ]moįƭJqR cDNA tɕ=5NS Ûŧ_Ǽā@mb˞ǰtː>6
RNA 10µL 5)PrimeScript Buffer 4µL PrimeScript RT Enzyme Mix I 1µL Random 6mers (100µM) 4µL RNase Free dH2O 1µL įˡ 20µL
£�¡� �£�� ÉÊ�¶· ÔßÜ Ä¾Éʾ½�¶· �2�
PCR `5ãZKX PCR �®º¢Å]£±y©ºtE52)PCR Master mix@mb Loading dye BĂȜJqR PerfectShot Ex Taq (Loading dye mix)��±�y~ɹ˗tɕ=RǔȚtɸM6 1) ×¾_˨ˉ@mb£±y©º���tȩįK5PCR ħƠtː>6
PerfectShot Ex-Taq 25µL cDNA 5µL Forward primer: NSL F3 (20µM) 1µL Reverse primer: NSL B3-6 (20µM) 1µL DNase/RNase free water 18µL įˡ 50µL
27
ħƠ ǫÙ �y�³Ǎ 1. Denaturation 94+ 3 Ă 1
Denaturation 98+ 10 ɽ Annealing 58+ 30 ɽ 2. PCR ħƠ Extension 72+ 45 ɽ
30
3. ǡʝ Extension 72+ 3 Ă 1 2) ×¾_˨ˉtȩįK5nested PCR ħƠtː>6
PerfectShot Ex-Taq 25µL 1st PCR ɔɉ 5µL Forward primer: F2 nest (20µM) 1µL Reverse primer: B2 nest (20µM) 1µL DNase/RNase free water 18µL įˡ 50µL
ħƠ ǫÙ �y�³Ǎ 1. Denaturation 94+ 3 Ă 1
Denaturation 98+ 10 ɽ Annealing 60+ 30 ɽ 2. PCR ħƠ Extension 72+ 45 ɽ
30
3. ǡʝ Extension 72+ 3 Ă 1 3) ȒȝĖ 1.5~2.0%w�µº��³TAE buffer âɕtɕ=5Mupid ª��³ȝĖ
ȁʊY 10µL _ PCR ɔɉtȝĖMp6|��z«¢µ©y�]mpdz˄Ƙ5
�±·�y³ª�º�ºYˌóǼāMp6ɡzy³�ÛŧBũŋKRŔ
į51st PCR _ŗƈɔɉZKX 256bp5nested PCR _ŗƈɔɉZKX 157bp_�·�B˪jnqp6 £�¡�¡��µÄ¾�ÉʾÆ�·¹�¶· TÚßá�eåäÿí ´¸ ��#ÛN��
NS Ûŧ_Ǽā]` One-Step Țtɕ=RřȚtɕ=pIZBYCp6Ǽß
]`<4-2-1-2>]ȳLXƵāKR RNA tɕ=p6
28
�
£�¡�¡� �w�
RT-PCR+ / [�VQIAGEN OneStep RT-PCR kit; QIAGEN�$] RNase Inhibitor [�:Recombinant RNase Inhibitor; MISPGH�] PCR+ / [�VTakara Ex-taqWMISPGH�] NS*�����QSGRU: 4-2-1-1A�?@DB £�¡�¡�¡�One-step RT-PCR ÔßÜ Nested PCR �2 ��B&">ECQSGRULNOF��?:RT-PCR��F#=;
RNase-Free dH2O 6.25 µl
5×Qiagen OneStep RT-PCR buffer 5 µl
5×Q Solution 5 µl
dNTP Mix 1 µl
Forward Primer NSL F3 (20 µM) 0.75 µl
Reverse Primer NSL B3-6 (20 µM) 0.75 µl
Qiagen One Step Enzyme Mix 1 µl
RNase Inhibitor (40U/µL) 0.25 µl
, RNA 5 µl
Total Volume 25µL
�� �� KGJT�
1. ('��� 509 30� 1
2. ('�+ �� 959 15� 1
Denaturation 949 30�
Annealing 589 30� 2. PCR��
Extension 729 1�
40
3. �! Extension 729 10� 1
��B&">ECQSGRULNOF��?:nested PCR��F#=;
dH2O 18.875 µl
10×Ex Taq buffer 2.5 µl
dNTP Mixture (2.5mM each) 2 µl
29
Forward Primer F2 nest (20 µM) 0.5 µl
Reverse Primer B2 nest (20 µM) 0.5 µl
Ex Taq (5U/µL) 0.125 µl
RT-PCR�� 0.5 µl
Total Volume 25µL
ȒȝĖ <4-2-1-4>Zİǿ]ȒȝĖtː=5Ûŧŗƈ_ɷ˪tː>6
�� �� KGJT�
1. Denaturation 949 3� 1
Denaturation 989 10�
Annealing 609 30� 2. PCR��
Extension 729 45�
25
3. �! Extension 729 3� 1
30
£�¢�� �eåäÿí ® ��#ÛN�ÔßÜ��#|J � ÁɚçíȇWHO]mUX5ɡzy³�_ÛŧŏĂ`5E1
Ûŧú_ sequence window Ő739bp58731-9469 Ý_ÛŧĄtĤ
ɃǶ_ÛŧĄZȑ˺˞ǰMpIZYː>IZBůjnqX=p 9)6ʾƋ
ǼßÅ_zy³�An sequence window Ő÷t»Ǝ]ŗƈJOpIZ
BńY<pIZBś=Rj5ÌǒɆ]ĂGX RT-nested PCR YŗƈJOR
_T5ÛŧĄ˞ǰ]ɕ=p6ZC]²w³�y« RT-PCR l�·¥·�
°�³ RT-PCR Y NS Ûŧ_ǼāBīʵY<URŔįYk5E1 Ûŧ_
Ɇǔ_ǒɆ<p=`ÃǒɆBŗƈYC\=IZB<p6P_Ŕį5ǼßÅ_
zy³�ÛŧBź\=IZBʮ?nqpRj5zy³�ĂtːURƘ5
Ă�·£³tɕ=X E1 Ûŧ_Ǽā@mbÛŧ˞ǰtː>Z˃=6 £�¢� �� �w � <4-2. �·¥·�°�³ RT-nested PCR ȚǾˋɨ˳NSÛŧ
ŗƈȚ>YâɕMp˨ˉ<4-2-1-1 ˨ˉ>tĤɃ_IZ6\@<4-2-2>_One-Step RT-PCR ȚYɕ=p˨ˉ`ěĂ\ƩƎBƚnq\=Rj5ǨȚ]`
J\=_Ó5×¾_˨ˉtɕ=p6 E1 Ûŧŗƈɕ£±y©º 5’ǒɆ E1-(2) ɕ £±y©
ºı
˪ ˯ Ő
bp
Ą
E1-2F 8633-8652 AGC GAC GCG GCC TGC TGG GG 1st PCR
E1-2R*1 9119-9138 CCA GCG CGT ATG TGG AGT CC
E1-6F 8664-8683 ACA CCG TGA TGA GCG TGT TC Nested PCR
@mb�º�
|·�·�
E1-10R*2 9110-9129 ATG TGG AGT CCG CAC TTG CG
*1: E1-2R _Õso] E1-2Rmix BâɕīʵY<p 10)6
E1-2Rmix: CCR GCG CGT ATG TGG AGY CC
*2: E1-10R _Õso] E1-10Rmix BâɕīʵY<p 10)6
E1-10Rmix: ATG TGG AGY CCG CAC TTS CG
ąW�! R: A/G, Y: T/C, S: G/CĆ
31
3’ǒɆ E1-(3) ɕ £±y©
ºı
˪ ˯ Ő
bp
Ą
E1-7F 8991-9010 TTG TGG GGG CCA CGC CAG AG 1st PCR
E1-12R 9521-9540 TGT GTG CCA TAC ACC ACG CC
E1-3F 9070-9089 CGG CGA GGT GTG GGT CAC GC Nested PCR
@mb�º�
|·�·�
E1-3R 9473-9492 ACC CGC GCG CTC GCG CGA TC
PCR ɔɉʔ˗����ã: QIAquick PCR Purification Kit; �w�·ɹ�� DNA �³Ƶā����ã: QIAquick Gel Extraction Kit; �w�·ɹ�� �º�|·�·�����ã: BigDye® Terminators Cycle Sequencing Kit;
w£±y��y~���«�ɹ� �
£�¢�¡�� ÉÊ�¶· ÔßÜ Ä¾Éʾ½�¶·�
� 4-2-1-3.YˮǎKR cDNA tɕ=p6ħƠǫÙ`×¾]ɸM6�
1) PCR ħƠ PerfectShot Ex-Taq 25µL cDNA 5µL 5’ǒɆ E1-(2) 3’ǒɆ E1-(3) Forward primer (20µM) E1-2F E1-7F 1µL Reverse primer (20µM) E1-2R E1-12R 1µL DNase/RNase free water 18µL įˡ 50µL
ħƠ ǫÙ �y�³Ǎ 1. Denaturation 94+ 3 Ă 1
Denaturation 98+ 10 ɽ Annealing 61+ 30 ɽ 2. PCR ħƠ Extension 72+ 45 ɽ
30
3. ǡʝ Extension 72+ 3 Ă 1
32
2) ×¾_˨ˉtȩįK5nested PCR ħƠtː>6
PerfectShot Ex-Taq 25µL 1st PCR ɔɉ 5µL 5’ǒɆ E1-(2) 3’ǒɆ E1-(3) Forward primer (20µM) E1-6F E1-3F 1µL Reverse primer (20µM) E1-10R E1-3R 1µL DNase/RNase free water 18µL įˡ 50µL
ħƠ ǫÙ �y�³Ǎ 1. Denaturation 94+ 3 Ă 1
Denaturation 98+ 10 ɽ Annealing E1-(2): 59+ 30 ɽ
E1-(3): 66+ 30 ɽ 2. PCR ħƠ
Extension 72+ 45 ɽ
30
3. ǡʝ Extension 72+ 3 Ă 1 3) ȒȝĖ 1.5%w�µº��³TAE buffer âɕtɕ=5Mupid ª��³ȝĖȁʊ
Y 10µL _ PCR ɔɉtȝĖMp6|��z«¢µ©y�]mpdz˄Ƙ5�±
·�y³ª�º�ºYˌóǼāMp61st PCR _ŗƈɔɉ` 5’ǒɆ E1-(2)Y506bp, 3’ǒɆ E1-(3)Y 551bp _�·�ZKX˪jnqp6»ǔ5nested PCR_ŗƈɔɉ` 5’ǒɆ E1-(2)Y 466bp, 3’ǒɆ E1-(3)Y 423bp Z_�·�ZK
X˪jnqp6 £�¢�¢��¶· b^Ûo{ØïäĀèòìĄèçĂí�2�
1) PCR ɔɉ_ʔ˗ � PCR ɔɉBȒȝĖYɷ˪YCR\na5PCR ɔɉ_ʔ˗tː>6ĝ»�
·�ZKX˪jnqRŔį]`Ƅ˰_ PCR ɔɉʔ˗���tɕ=pZʒæ
Y<p6�·�B˘Ǎ˪jnqRŔį]`5�³Anɭɪ_�·�_htă
oāKXʔ˗Mp6�º�|·�£±y©º`5Ƈ nested PCR ]âɕK
33
R£±y©ºtâɕMpIZBāǬp M\sT55’ǒɆ E1-(2)_Ŕį5E1-6FgR` E1-10R53’ǒɆ E1-(3)_Ŕį5E1-3F gR` E1-3R ņ 6£±
y©ºE1-3R tɕ=R�y´���º�|·�ħƠY`5țƖBCq=]ā
N5ěĂ\�º�tƚnq\=IZB<p6P_Ŕį`5£±y©ºE1-S4R: CGG CGG TGA CGA ACT TCC ą9405-9422 ŕŒŐ]ŷƠ tɕ=X˞ǰ
tː>6 2) �º�|·�ħƠ � ×¾_˨ˉ@mb£±y©ºtȩįK5�y�³�º�|·�ħƠtː>6
H2O 10µL Primer (3.2µM)* 1µL BigDye Terminator Ready Reaction Mix 8µL** ʔ˗ PCR ɔɉ5-20ng/µL 1µL
*Primer: 5’ǒɆ E1-(2)_Ŕį5E1-6F gR` E1-10R53’ǒɆ E1-(3)_Ŕį5
E1-3F gR` E1-3R<p=` E1-S4Rtɕ=p6 **BigDye Terminator Ready Reaction Mix`)5 BigDye sequencing bufferYƆīʵY<pB5ˀƆɎ`ĮʿYǼˢtː>Ɵ˚B<p6
ħƠǫÙ �y�³Ǎ 96+ 1 Ă 1
96+ 10 ɽ 50+ 5 ɽ 60+ 4 Ă
25
3) �º�|·�ħƠɔɉʔ˗@mb�º�|·�˞ǰ AutoSeq G-50GE ¤³��wʊtɕ=5ǦħƠ�y�ºª�º�ºt
ģMp6I_ʔ˗ɔɉt DNA �º�|·�º]mo˞ǰK5ÛŧĄ_
Ȗůtː>6 £�¢�£�� ìĄèçĂísKÛ|J�
� 5’ǒɆ@mb 3’ǒɆ_ÛŧĄƥœtV\DįsOX5739bp An\p
ÛŧŏȖůŐ8731-9469 Ý_ĄƥœtǾʑMpņ 6
34
ņ� ÛŧŏȖůŐsequence windowĸ˽_ cDNA Ą AÛŧŏȖůŐŗƈɕ£±y©º_˪˯Ý_ȃƑņ5 B
TO-336.GMK5 ǶTO-336 ¶��·Ƕ_˜Ƕ5Ûŧŏ 1a5accession number: AB588192_ĄtɸM6E1-(2)ǒɆ@mb E1-(3)ǒɆŗƈ£±
y©ºʟįÝt5PqQq˴@mbŨYɸM6ÛŧŏȖůŐ_ǡą
ZǡƘ_ 10 ŕŒNVtşŨYɸM6 � ɐŋ WHO BÅƞZ\UX5Įň_ŰűAnœķJqRɡzy³�
35
ÛŧƥœtkZ]ÛŧŏĂBːsqX=p 11)6ɐŋ_ZIrɡzy³
�`V_ Clade ]ŝCEĂGnqpIZBĆǚKX=p6Jn] Clade 1]` 10 V_Ûŧŏ1a, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1JB5Clade 2]`V_Ûŧŏ2A, 2B, 2CBũŋMp6w³¡v¥��_ŹǏŨY
˓ˣJqRÛŧŏ`Øprovisional_ÛŧŏY<p6WHO ]mUX
ůjnqRPqQq_Ûŧŏ_ĤɃǶ_Ą˓Z<sOXĂŧʖʠȅ
˞ǰtː>IZ]mo5ǼāJqRzy³�B[_Ûŧŏ]ĂJqp_
ABĆůYCpņ 6˿Ɗ5Ûŧŏ 1a zy³�`¶��·zy³�t
=XeZu[ǼāJqX=\=6I_RjǨÛŧŏzy³�BǼāJq
RŔį]`¶��·ǶY<pīʵƢBE5˪jnqpɤɋ`¶��·ǁʂ
_čħƠBɟsqp6¶��·ǁʂ_čħƠ_Ŕį]`ɡZ`˦ǒK\=6
gRÁɚĮňAnœķJqRĄ�º�tɕ=XĂŧɠūɪ\˞ǰBː?5
ǼāJqRzy³�]mpƩdzB˼öãAijAtĆćMpIZ\[BīʵZ
\p6 2013 Ɗ_ɡĹıȚ_NJů 11)Y`5IqgYØÛŧŏY<UR 1H, 1I, 1JBȊƑ\Ûŧŏ]ǙǸKR6đ?X»_ĤɃǶ_ǞǓ1H _»Ƕ@m
bıɾ_ǎɒB\JqR˓ 6 E1 Ûŧ˞ǰB\JqRŔį5 Ûŧŏƥœ]đ?5 ÛŧĄ]V=Xk
NESID ɣġßǼāƥœ���«]ɧtː>6NS ÛŧBǼāJqRk_
_5E1 Ûŧ˞ǰBāǬ\AURŔį]`5ŏć¿ǚnot typedZKX
NESID ɣġßǼāƥœ���«]ɧtː>6 gR5īʵ\o DDBJ ʊ_Ûŧ�º��·�c_ɧkː>IZBǂš
Jqp6
36
˓� ɡzy³�ĮÛŧŏ_ĤɃǶ
Û
ŧŏ
ĤɃǶ_Ǔıɾ
ĤɃǶ_ċıɾ
Ǩ©�®w³ʉÌɇˣ˻
Accession
Number
RVi/Brussels.BEL/0.63/(VAC) RVi/BEL/63[1a]VAC AF188704
RVi/New Jersey.USA/0.61/(VAC) RVi/NJ.USA/61[1a]VAC M30776
RVi/Pennsylvania.USA/0.64/(VAC) RVi/PA.USA/64[1a]VAC L78917
1a
RVi/Toyama.JPN/0.67/ Rvi/Toyama.JPN/67[1a] AB047330
RVi/Jerusalem.ISR/0.75/ RVi/Jerusalem.ISR/75[1B] AY968207
RVi/Tiberias.ISR/0.88/ RVi/Tiberias.ISR/88[1B] AY968209
1B
RVi/Bene Berak.ISR/0.79/ RVi/BeneBerak.ISR/79[1B] AY968208
RVi/California.USA/0.91/ RVi/Los Angeles.CA.USA/91[1C] AY968212
RVi/San Salvador.SLV/0.02/ RVi/SLV/02[1C] AY968211
1C
RVi/Panama City.PAN/0.99/ RVi/PAN/99[1C] AY968217
RVi/Tokyo.JPN/0.90/ Rvi/Tokyo.JPN/90CRS[1D] AY968214 1D
RVi/Saitama.JPN/0.94/ Rvi/Saitama.JPN/94[1D] AY968216
RVi/Shandong.CHN/0.02/ RVi/Dezhou.CHN/02[1E] AY968210 1E
RVi/Kuala Lumpur.MYS/0.01/ RVi/MYS/01[1E] AY968221
RVi/Shandong.CHN/0.00/ Rvi/Linqing.CHN/00[1F] AY968213 1F
RVi/Anhui.CHN/0.00/ RVi/Dangshan.CHN/00[1F] AY968215
RVi/Kampala.UGA/20.01/ Rvi/UGA/20.01[1G] EF588978
RVi/Ontario.CAN/27.05/ Rvi/Ontario.CAN/27.05[1G] EF588970
1G
RVi/Minsk.BLR/29.04/ RVi/Minsk.BLR/29.04[1G] AM258945
RVi/Minsk.BLR/28.05/2 Rvi/Minsk.BLR/28.05[1H] AM258953
RVi/Ryazan.RUS/09.08/ HG326276
1H
Rvi/Novokuznetsk.RUS/04[1H] EF421977
RVi/Milan.ITA/46.92/ Rvi/Milan.ITA/46.92[1I] AY161360 1I
RVi/London.GBR/0.86/ Rvi/London.GBR/86[1I] AF039122
RVi/Kagoshima.JPN/22.04/ Rvi/Kagoshima.JPN/22.04[1J] AB285129 1J
RVi/Miyazaki.JPN/10.01/ Rvi/Miyazaki.JPN/10.01[1J]CRS AB285130
RVi/Beijing.CHN/0.79/ RVi/Beijing.CHN/79[2A] AY258322 2A
RVi/Beijing.CHN/0.80/(VAC) RVi/Beijing.CHN/80[2A]VAC AY258323
37
RVi/TelAviv.ISR/0.68/ RVi/TelAviv.ISR/68[2B] AY968219
RVi/Washington.USA/16.00/ Rvi/Seattle.WA.USA/16.00[2B] AY968220
2B
RVi/Anhui.CHN/0.00/2 RVi/Anqing.CHN/00/2[2B] AY968218
RVi/Moscow.RUS/0.67/ Rvi/Moscow.RUS/67[2C] DQ388279 2C
RVi/Moscow.RUS/0.97/ Rvi/Moscow.RUS/97[2C] DQ085340
38
�
ņ� ɡzy³�ĤɃǶ@mbǗǨ_¶��·Ƕ]mpʖʠȅ E1 Ûŧ_ÛŧŏȖůŐ739bp_Ąƥœtɕ=X˿ǁįȚ]m
UX˞ǰtːUR6ɐŋ 13 ʂ_Ûŧŏ]ĂJqX=p6ǗǨ_¶��·
ǶTO-336 Ƕ5ǮȣǶ5ȆǶ@mb÷ÁɚYÆ]âɕJqp¶��·Ƕ
RVi/Pennsylvania.USA/0.64/(Vac), ćı RA27/3 Ƕ`Ûŧŏ 1a ]Ă
RVi/Brussels.BEL/0.63/(VAC)
Takahashi(VAc;Kitasato)
RVi/New Jersey.USA/0.61/(VAC)
1a
RVi/San Salvador.SLV/0.02/
RVi/California.USA/0.91/
RVi/Panama City.PAN/0.99/
1C
RVi/Toyama.JPN/0.67/
TO-336(Vac;Takeda)
Matsuura(Vac;Biken)
RVi/Pennsylvania.USA/0.64/(VAC)
1a
RVi/Tiberias.ISR/0.88/
RVi/Bene Berak.ISR/0.79/
RVi/Jerusalem.ISR/0.75/
1B
RVi/Minsk.BLR/29.04/
RVi/Ontario.CAN/27.05/
RVi/Kampala.UGA/20.01/
1G
RVi/Ryazan.RUS/09.08/
RVi/Minsk.BLR/28.05/2/1H
Rvi/London.GBR/0.86/
RVi/Milan.ITA/46.92/1I
RVi/Tokyo.JPN/0.90/
RVi/Saitama.JPN/0.94/1D
RVi/Kuala Lumpur.MYS/0.01/
RVi/Shandong.CHN/0.02/1E
RVi/Miyazaki.JPN/10.01/
RVi/Kagoshima.JPN/22.04/1J
RVi/Shandong.CHN/0.00/
RVi/Anhui.CHN/0.00/1F
RVi/Beijing.CHN/0.80/(VAC)
RVi/Beijing.CHN/0.79/2A
RVi/TelAviv.ISR/0.68/
RVi/Washington.USA/16.00/
RVi/Anhui.CHN/0.00/2/
2B
RVi/Moscow.RUS/0.97/
RVi/Moscow.RUS/0.67/2C
0.01
39
Jqp6 £�£�� þãÿîäú ·¹�¶· ÔßÜêĂøĂìüóÿ ·¹�¶· Û�3êĂò
āĄÿ�
½ˣ RT-PCR ]âɕMpƢ�·�µº³`ňʅƩdzɤɶʄƮzy³�ʉ¼
ʉÌűYƅKX=p6 ¤�� åäÿí���
zy³�_Ă@mbİů]` 3;t˚Mp6 ¤� �� �cqu�
śE_ʜʴBɡzy³�ƩĪƢY<pB5Vero5VeroE6 @mb RK-13 ʜ
ʴBňúŚY_ŰʨBś=6\@5ʾƋɤɋʊmoɡzy³�ƩdzZęć
Bń\Ŕį`5ɡzy³�_ĂǔȚ]ĊK5�� SLAM ɦɐ Vero ʜ
ʴtɕ=XĂtː>IZkīʵY<p6gR5ɡzy³�_ŗȌ`Ƴɲ
ˆČ_¡v·��·]mUXƲĉJqp_Y5zy³�Ă_Rj_őȨ
]`đ?\=ǔBm= 12)6 ¤�¡�� ��AT�
×¾`ňʅƩdzɤɶʄƮYŰǕKX=pǔȚRK-13 ʜʴâɕtkZ]ˣ
˻MpB5ʜʴʂ]mpƃlİLʜʴYkˀőŌ_ǫÙBɞ\p_Y5Į
ǼǴű]X˩ʜ`ǼˢMpIZBǤgK=6 %ɈˎȫBStȪđKR Eagle’s MEM ]ȤJOR RK13 ʜʴ
3)10cells/mlt£±����¡±��12.5cm2]»Ǩ<Ro
4ml5kKE` 6 ʃ£´º�»ʃƕRo 3mL tLJʂK537+_ȿ�
�őłY 2 ǗőMp6 0.2ml _ǼßtǁʂMp6űȯY 1 ǜĵɳKRƘ5ǁʂǩǑt_Q
AN] 2%BStĴiőȨt 4mlđ? 35~37+_ȿ��őłYő
Mp35+_ǔBǤgK=B 37+YkĂīʵY<p 6ǼßBŻ
_Ŕį`ǁʂǩǑt=RƘ]őŌtđ?pŻ`ʜʴc_ȐƢBƔ
=Rj 6ʭǗ]ʜʴřƭĕDz(CPE)BhnqRŔį`�·£³_Ɋ
ɞɪ\ȐƢ]mpZʮ?nqp_YǓK=őȨ]ÏDžMp6ǁʂƘ
40
4 Ǘɭ]őȨtÏDžK5ǁʂƘ 7 ǗɭgYőtʢGp6ƇI_
ȍY`zy³�]mp CPE `hnq\=6 ƩdzʜʴtőȨZZk]þʟˍ˞K5P_»(1ml)tǓK=ʜʴ]
ǁʂK5İǿ]őMp6ƇI_ʡÕőtŃʩoMB5CPEB˝ŶYCpm>]\qaŃǍtȭnMIZBYCp6őʝÉƘʜ
ʴtþʟˍ˞K5P_Ƙ 3000rpm520 Ă_ƞ½ȫtĂ˨ǑZK
zy³�İůtː>6»˂]ɡzy³�]mpCPE`Ɠ=RjCPEB˝ŶYC\EZkĂ˨ǑÅ]ɡzy³�BũŋKX=pīʵƢ
BěĂ]<p6gRɡzy³�]mp CPE `ɊƝB\=_Y CPE˝Ŷ]mpĂİů_Ćů`ËŰ½ńY<p6
¤�¢�� �&AT�
ɡzy³�_İů]`zy³�ÛŧǼāRT-PCR 5ˌóƳßȚ]mp
zy³�ƳġǼā5Ƴˎȫ]mpÅĺȚʊB<pB5ÅĺȚ`ǜBAAp
IZlÅĺƳß_ɷç\[ƯBAApRjǂšK\=6Ǩ©�®w³Y`
ÛŧǼāȚ ǖ @mbˌóƳßȚ]mpƳġǼāȚtˣ˻Mp6\@5
ĆůgY_ǜBɵE5Ăŧɠūɪ\˞ǰkīʵZ\pzy³�ÛŧǼā
tʉ»ƴZKXâɕMpIZBǂšJqp6 ¤�¢� �� y�9�T�
âɕʜʴ¹˨ˉʊ`zy³�ĂZİǿ_k_tâɕMpB5ʜʴ_ő`
�±�˗�·�º�±y�8 �·�ºʊYː>6 1) RK-13 ʜʴt�·�º�±y�YőK5Ă˨Ǒ 25*l tǁʂK
35~37+Y 1 ǜNjʪƘ5őȨt 0.4ml đ? 35~37+_ȿ��ő
łúYőMp 2) ƩdzƘ CPE BɐqRnʘ 3-4 Ǘ5CPE B˪jnq\=ŔįYk 5-7
ǗɭY 5ʜʴt PBS YȟȢKRƘ54%�±§³«w³���Y 30 Ă
ŇůMp6PBS YȟȢƘ50.5%TritonX-100 đ PBS tűȯ 15 ĂYƩ
àK5ʽĀɒtMp6 3) ŇůKRʜʴ]»ȈƳßZKXƄ˰JqX=pƳɡzy³�ʔ˗©z
�¬��µº�³Ƴß ã abcam ab34749, USBiological R9700-05A
t 1;2*g/mL ]ƆK5»ęə<Ro 100~200*l đ?5çȱʎ]öq
41
37+1 ǜĀɒMp6 4) PBS Y 5-6 ŃȟȢƘ5FITC Ȃ˯Ƴ©z� IgG Ƴß ȻƎ]kmpB 1
300;500 Ɔt 100~200*l _Oçȱʎ]öq 37+1 ǜħƠJOp 5) PBS Y 5-6 ŃȟȢƘ5ˌó˝ŶɕŸöČt_OX��º�±�t@C5
ˌóƜY˝ŶMp6İǿ]ĀɒKRƩdzʜʴtŷɃZK5ɡz
y³�]Ɋɞɪ\ˌóBǼāJqpŔįtɡzy³�ƢZĆůMp6
¥�� N�åäÿíÛ��T�
WHO ]mUX5ɡzy³�ŚǶ_ĹıǔȚB×¾_o]ɸJqX=
p 11)62013 Ɗ_NJˠYƊ_ƥœˣ˻ʊB»řǞJqR6 1)ʾƋǩǑAnʜʴő]moĂJqRzy³�Ƕ RVi/City.Country/Weeks.Year/isolation number[Genotype] 2)ʾƋǩǑɗǬ RNA AnÛŧBǼāJqRzy³�Ƕ RVs/City.Country/Weeks.Year/isolation number[Genotype] CityʾƋǼß_ƚnqRƍɱıgR`njÖƺůƄı6njÖƺůƄ
ı_ǜ` C(city) tVGp6 CountryʾƋǼß_ƚnqRňı_ɜɾǗǨ_Ŕį JPN Weeks.Yearɦɤɠū@mbɦɤƊtˣ˻Mp6ɡ_Ŕį5ɦɡ
ZɦɄ`efİǜ]āɐMpB5=NqA_ɤɋKA˪jnq\=Ŕįl¿
Ƣ_ŔįB<p6gR5ɦɤǗgR`ɤɋ_ʂ_ƥœBƚnq\=IZ
B<p6P_Rj5ɦɤƊZKX`×¾_]ðòƎ_=Ǘǜt˵ɀ
ZKXˣ˻Mp6�
.ɦɡāɐǗ�
/ɦɡB˪jnq\=ŔįkKE`ɤɋ_ʂƥœBƚnq\=Ŕį5\u
nA_ɦɤ_āɐǗ�
0ɦɤǗ_ƥœBƚnq\=Ŕį5ǼßǀĩǗ6�
\@5Ɗƥœ`5˙ǝ_¾Ìǹ_htˣ˻Mp6�
Isolation numberƤʯ_ɦɓKRƄ@mbɦɤBİ»_zy³�ǶB
˘ǍũŋMpŔį51 An]ǎɒɝĭtˣ˻~£�°·
42
GenotypeÛŧŏ òŞƢɡɤëʬCRSƤʯ5òŞƢɡƩdzCRIʯ_ʾƋǩǑAn
ƚnqRzy³�Ƕ]`[Genotype]_Ƙr]PqQq(CRS)5(CRI)Zˣ˻M
p6ÃʯZk City, Country ]`āɓŌ5Weeks, Year ]`āɓǗǼßǀ
ĩǗY`\EtɕMp6 ¶��·Ƕ]`[Genotype]_Ƙr](VAC)Zˣ˻Mp6 ãŰŋMpk_Y`<ogOu ã1998 Ɗ 3 ɭ]ǭÐYɦɤKRƤʯAnƚnqRzy³�ǶY5
İ»_Ƥʯ_>Tɝɭ]˞ǰJqRk_6zy³�BĂJqX@o5
Ûŧŏ` 1D6 RVi/Tokyo.JPN/03.98/2[1D] ã 2009 Ɗ 28 ɭ]ɻƀƄYāɓKR CRS ƤʯɗǬ_zy³�6zy
³�ÛŧBǼāJqX@o5Ûŧŏ` 1B6 RVs/FukuokaC.JPN/28.09/[1B](CRS) 7. �����1) ĠɓĔïɰķɸʉɩÌěÌĭKu]MpɊůƩdzɤÊƺ5Ɖƭ
26 Ɗ 3 Ǣ 28 Ǘ 2) ƉġĬȅeAɡȡː@mbòŞƢɡɤëʬ_ɦɓƲĉ]Mpʥơ
DŽ˟6ĠɓĔïɼūɶʄ˲˖ē� Ǔˁ¹ûˁƩdzɤɶʄËǽĂƶɶʄ
ɏ� ɡȡː]Zk\>ȎŧƩdz_ÊŷʋǾʑ]Mpɶʄ52004. 3) World Health Organization: Manual for the laboratory diagnosis of
measles and rubella virus infection. Second edition. WHO/IVB/07.01. 2007.
4) ȽėĿeAɡ˦ǒ©�®w³ʉɇ 52015. 5) ňʅƩdzɤɶʄƮzy³�ʉ¼Ʃdzɤɠū�·�ºKuƳßå_
Džʌ˭hǠ?]MpǼˢ5ňʅƩdzɤɶʄƮ§º«¦º�52014Ɗ 6 ǢNJˠɇ
6) đˊ˅Ūɡ HI ƳßåȰů_ˑƑ6ʾ ƋZzy³�519(2)5127-13051991.
43
7) đˊ˅ŪˎȫĀɒɕ�~²·_Āɒ`Ɵ˚ǫÙY`\=6ʾƋZzy
³�522(2)5S7551994. 8) Inouye, S.: Micro-modification of kaolin treatment of serum for the
rubella hemagglutination-inhibition test. J. Med. Microbiol., 9, 501-502, 1976.
9) World Health Organization: Standardization of the nomenclature for genetic characteristics of wild-type rubella viruses. Wkly. Epidemiol. Rec., 80(14), 126-132, 2005.
10) Yasui et al.: Detection and genotyping of rubella virus from exanthematous patients suspected of having measles using reverse transcription-PCR. Jpn. J. Infect. Dis., 67, 389-391, 2014.
11) World Health Organization: Rubella virus nomenclature update: 2013. Wkly. Epidemiol. Rec., 88(32), 337-348, 2013.
12) Umino, Y and Tashiro, M.: Inhibition of rubella virus growth by Fungizone. Vaccine, 19, 1369-1372, 2001.
��������
¹÷ňƍɱ/njÖƄ˒ɓɶʄƮ�
¹ňʅƩdzɤɶʄƮzy³�ʉ¼�
:208-0011ǭÐȋˈǪſƄūʼn�4-7-1
Tel:042-561-0771
Fax:042-565-3315
�
��� ����
� ʉ»ɇƉƭ 14 Ɗ 3 Ǣ � ʉÌɇƉƭ 24 Ɗ 12 Ǣ � ʉ¼ɇƉƭ 27 Ɗ 3 Ǣ � ʉ 3.1 ɇƉƭ 27 Ɗ 8 Ǣ � ʉ 3.2 ɇƉƭ 29 Ɗ 8 Ǣ²w³�y« RT-PCR ˨ǡĘ]@GpĤ
Ƀ RNA Ɔȍ_řǞ �
�