
Á÷¸Ð²¡¶¾¿ìËÙ¼ì²â·½·¨ÌØÒìÐÔºÍÃô¸ÐÐÔÑо¿
·¢²¼Ê±¼ä£º2015/2/10 13:07:50
Á÷¸Ð²¡¶¾¿ìËÙ¼ì²â·½·¨ÌØÒìÐÔºÍÃô¸ÐÐÔÑо¿
ÓÈ·ïÐË1 , ¾ÓÀöö©2 , Õž´Æ½1 , Ê©¡¡Ç¿2 , Âí¹ãÔ´1 , ¼ªÐÓÉú1 , Îâ¼ÒÁÖ1 , Á衡ϼ1
(1. ÎÞÎýÊм²²¡Ô¤·À¿ØÖÆÖÐÐÄ, ½ËÕÎÞÎý214023; 2. ¸´µ©´óѧ¹«¹²ÎÀÉúѧԺ, ÉϺ£200032)
¡¡
¡¡ ÕªÒª:Ä¿µÄ¡¡±È½Ï¶àÖØÄæ×ªÂ¼¾ÛºÏøÁ´·´Ó¦(mRT2PCR)ºÍ½ºÌå½ð·¨¼ì²âÁ÷¸Ð²¡¶¾µÄÃô¸ÐÐÔºÍÌØÒìÐÔ¡£·½·¨²ÉÓÃmRT2PCRºÍ½ºÌå½ð·¨¼ì²â¾Ï¸°ûÅàÑøºÍºìϸ°ûÄý¼¯ÒÖÖÆÊÔÑé(H I)¼ø¶¨ÎªA /H1¡¢A /H3¡¢BÐÍÁ÷¸Ð²¡¶¾µÄ±ÇÑÊÊñ걾78·Ý,ÒõÐÔ±ÇÑÊÊñ걾40·Ý,²¢¶ÔÒÑÏ¡ÊͳÉ101 ¡«105²¡¶¾°ëÊý¸ÐȾÁ¿( TCID50) /0. 1 mLµÄ3ÖêH1¡¢H3¡¢BÐÍÁ÷¸Ð²¡¶¾½øÐÐÃô¸ÐÐÔÊÔÑé¡£½á¹û¡¡mRT2PCR¶ÔAÐÍÁ÷¸Ð²¡¶¾µÄ¼ì²âÃô¸ÐÐÔΪ44. 8% ,ÌØÒìÐÔΪ97. 5%;¶ÔBÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ20. 0% ,ÌØÒìÐÔΪ100. 0%¡£½ºÌå½ð·¨¶ÔAÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ
44. 8% ,ÌØÒìÐÔΪ100. 0%;¶ÔB ÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ5. 0% ,ÌØÒìÐÔΪ97. 5%¡£¶Ô¾20 ´úÄÚ¹·Éöϸ°û(MDCK)·ÖÀëÅàÑøÑôÐÔµÄ4·ÝA /H1¡¢4·ÝA /H3ºÍ3·ÝBÐÍÁ÷¸ÐÅàÑøÒº,mRT2PCRºÍ½ºÌå½ð·¨¼ì²âµÄÑôÐÔ·ûºÏÂÊÓëÐͱð·ûºÏÂʾùΪ100. 0%¡£mRT2PCR¼ì²âÁ÷¸Ð²¡¶¾Ãô¸ÐÐÔΪ103 TCID50 /0. 1 mL;½ºÌå½ð·¨¼ì²âµÄÃô¸ÐÐÔΪ103 TCID50 /0. 1 mL¡£½áÂÛ¡¡mRT2PCRºÍ½ºÌå½ð·¨µÄÃô¸ÐÐÔÓëÌØÒìÐÔÎÞ²îÒì; mRT2PCRºÍ½ºÌå½ð·¨¾ù¿ÉÓÃÓÚÈËȺÁ÷¸Ð±ÇÑÊÊñ걾Á÷¸Ð²¡¶¾¿ìËÙ¼ì²â,ÌØ±ðÊǾۼ¯ÐÔ±©·¢Á÷¸Ð»¼Õß,½¨Òé²ÉÑùʱ±ê±¾ÊýÒªÊʵ±·Å´ó2¡«3±¶¡£mRT2PCR¿É½øÒ»²½È·¶¨Á÷¸Ð²¡¶¾µÄH1ºÍH3ÑÇÐÍ¡£
¹Ø¼ü´Ê:Á÷¸Ð²¡¶¾;·ÖÀë;¶àÖØÄæ×ªÂ¼¾ÛºÏøÁ´·´Ó¦;½ºÌå½ð·¨
Research on the specificity and sensitivity of rapid detection methods of influenza virus¡¡YOU Fengxing1 ,JU L iwen2 , ZHANG J ingping1 , SHI Q iang2 , MA Guangyuan1 , J I X ingsheng1 , WU J ialin1 , L ING X ia1. ¡¡( 1. W uxiCenter forD iseases Prevention and Control, J iangsu W ux i 214023,
¡¡¡¡Abstract: Objective To research the sensitivity and specificity of multip lex reverse transcrip tion2polymerasechain reaction (mRT2PCR) and colloidal gold rap id test for detection of influenza virus. Methods¡¡The samp les beingidentified as influenza virus A /H1, A /H3 and B by cell culture and hemagglutination inhibition (H I) test weredetermined bymRT2PCR and colloidal gold rap id test in 78 positive samp les and 40 negative samp les of nasopharynxswabs. Additionally, the sensitivitywas determined in the samp les of influenza virus H1, H3 and B diluted from 101 to105 TCID50 /0. 1mL. Results¡¡The sensitivity of mRT2PCR to influenza virus A was 44. 8% , and its specificity toinfluenza virusA was 97. 5%. The sensitivity of mRT2PCR to influenza virus B was 20. 0% , and its specificity toinfluenza virus B was 100. 0%. The sensitivity of colloidal gold rap id test to influenza virus A was 44. 8% , and its specificity to influenza virusA was 100. 0%. The sensitivity of colloidal gold rap id test to influenza virusB was 5. 0% ,and its specificity to influenza virusB was 97. 5%. mRT2PCR and colloidal gold rap id test detected 4 samp les ofA /H1,4 samp les of A /H3 and 3 samp les of B being identified byMDCK cell culture, and the positive coincidence and typecoincidence of the two testswere 100. 0%. The sensitivity ofmRT2PCR to influenza viruswas 103 TCID50 /0. 1 mL, andthe sensitivity of colloidal gold rap id test to influenza virus was 103 TCID50 /0. 1 mL. Conclusion s¡¡There is no
statistical difference in the sensitivity and specificity of mRT2PCR and colloidal gold rap id test. Both mRT2PCR andcolloidal gold rap id test can be used in rap id detection of nasopharynx swabs samp les of influenza virus, especially in thecluster of patients with exp losive influenza virus, but the quantity of samp le should be increased 2 to 3 times in thecourse of samp ling. The tests ofmRT2PCR can identify subtype H1 and H3 of influenza virus.
Key words: Influenza virus; Isolation; Multip lex reverse transcrip tion2polymerase chain reaction; Rap id test,colloidal gold
ÒýÆðÁ÷ÐÐÐÔ¸Ðð(¼ò³ÆÁ÷¸Ð)µÄÖ÷Òª²¡¶¾ÊÇA¡¢BÐÍÁ÷¸Ð²¡¶¾,ͨ³£Ö»ÓÐAÐͺÍBÐÍÁ÷¸Ð²¡¶¾»áÒýÆð´óÁ÷Ðлò¾Ö²¿µÄ±©·¢Á÷ÐÐ[ 1 ] ¡£´«Í³²¡¶¾·ÖÀëÅàÑøºÍºìϸ°ûÄý¼¯ÒÖÖÆÊÔÑé(H I)¼ø¶¨·½·¨ÌØÒìÐÔºÍÃô¸ÐÐÔÇ¿,µ«²¡¶¾·ÖÀëÅàÑøºÄ·Ñʱ¼ä½Ï³¤,ÐèÒªÓÐÁ÷¸Ð²¡¶¾ÐÍÌØÒìÐÔ¿¹ÑªÇå,¶ÔÓÚÈËȺ¾Û¼¯ÐÔÁ÷¸Ð±©·¢Ìṩ²¡ÔÌåÕï¶ÏÒÀ¾ÝÏà¶Ô½ÏÂý¡£½ºÌå½ð·¨³ö½á¹û¿ìËÙ,Ö»Ðè20 min,²Ù×÷¼ò±ã,ÊÊÓÚÏÖ³¡¼ì²â,µ«ÆäÖ»Äܼø¶¨AÓëBÐÍÁ÷¸Ð,Ŀǰ»¹²»ÄܽøÐÐÑÇÐͼø¶¨,²¢ÇÒûÓйú²úÊÔ¼Á,ÊÔ¼ÁºÐ¼Û¸ñ°º¹ó,ÓÐÎÄÏ×±¨µÀÃô¸ÐÐÔÏà¶Ô½ÏµÍ[ 2 ] ¡£¶àÖØÄæ×ªÂ¼¾ÛºÏøÁ´·´Ó¦(mRT2PCR)¼ì²â¹ý³ÌÐè4 h,²¢ÐèÒªÒ»¶¨µÄÒÇÆ÷É豸,Ó뽺Ìå½ð·¨±È½Ï³É±¾µÍ,²¢ÇÒ¿ÉÒÔ½øÐÐÑÇÐͼø¶¨¡£¶ÔÓÚ¾Û¼¯ÐÔÁ÷¸Ð±©·¢,Ê×ÏÈÒª¿ìËÙ³ö½á¹û,²¢ÇÒ×îºÃÄܹ»ÌṩÐÍÓëÑÇÐ͵ÄÕï¶Ï¡£»ùÓÚ´Ë,ÎÒÃǶԽºÌå½ð·¨ºÍmRT2PCRÔÚÁ÷¸Ð²¡¶¾µÄ¿ìËÙ¼ì²â½øÐÐÁ˽ÏÎªÈ«ÃæµÄ±È½ÏÑо¿¡£
²ÄÁϺͷ½·¨
Ò»¡¢ÊÔÑé²ÄÁÏ
1. ±ê±¾ÓëÀ´Ô´¡¡±ê±¾À´×ÔÎÞÎýÊÐÈËÃñÒ½Ôº¶ù
ͯҽԺ·Ö²¿ºÍÎÞÎýÊеÚÈýÈËÃñÒ½Ôº¶ùÄڿƹú¼ÒÁ÷
¸Ð¼à²âÍøÂçÉÚµãÒ½Ôº,´Ó¾ÍÕïÁ÷¸ÐÑù²¡Àý¼°·ÎÑײ¡Àý(ÌåΡÝ
2. ÊÔ¼Á¡¡RNAÌáÈ¡ºÍmRT2PCRʹÓñ¦ÉúÎï
¹¤³ÌÓÐÏÞ¹«Ë¾Takara RNAiso PlusÊÔ¼ÁºÐ,ÅúºÅΪ
DV
À©Ôö²úÎï·Ö±ðΪ431¡¢210ºÍ390 bp¡£½ºÌå½ð·¨ÊÔ
¼ÁºÐÓɺ¼ÖÝ´´ÐÂÉúÎï¼ì¿Ø¼¼ÊõÓÐÏÞ¹«Ë¾Ìṩ(¹ú
Ò©×¼×ÖS20050106 ) ,ÅúºÅΪFA080301¡£ÒÇÆ÷Ϊ
ÃÀ¹úB IO2RAD Mycycler PCRÒÇ¡£
¶þ¡¢ÊµÑé·½·¨
1. ²¡¶¾·ÖÀëÅàÑøÓë¼ø¶¨¡¡²ÉÓÃ20´úÄÚ¹·Éö
ϸ°û(MDCK)·ÖÀëÅàÑø¡£±ê±¾Ô¤´¦Àí: 3 mL±ê±¾
¼Ó150¦ÌL¡°ËÄ¿¹¡±2¡«8 ¡æ±ùÏä¹ýÒ¹¡£½«Òѳ¤³Éµ¥
²ãµÄMDCKÓú¬0. 02 mL /mL¡°ËÄ¿¹¡±»ù´¡ÅàÑø»ù
Ï´2´Î,½ÓÖÖ150¡«300 ¦ÌL ±ê±¾,
2 hºó, ÎüÆú±ê±¾Òº, ¼ÓÅàÑøÎ¬³ÖÒº(ÖÕŨ¶ÈΪ
5¦Ìg/mLÒÈø¡¢¸÷50¦ÌL /mL¡°ËÄ¿¹¡±¡¢0. 2%СţѪÇå°×µ°°×) ,ÖÃ
ÄýÑôÐÔÅàÑøÒºÓùú¼ÒÁ÷¸ÐÖÐÐÄͳһÏ·¢µÄÒÑÖªÑÇÐÍѪÇå×÷H I,¼ø¶¨Ëù·ÖÀëÁ÷¸Ð²¡¶¾µÄÐͱð¡£
2. ½ºÌå½ð·¨¼ì²â¡¡°´ÕÕÊÔ¼ÁºÐ˵Ã÷Êé²Ù×÷²½
Öè½øÐС£
3. Takara RNAiso Plus RNAÌáÈ¡¡¡°´´óÁ¬±¦
ÉúÎïTakaraÊÔ¼ÁºÐÌáÈ¡¡£
4. mRT2PCR¡¡°´ÕÕTakaraÊÔ¼ÁºÐ˵Ã÷Êé²Ù×÷²½Öè½øÐС£mRT2PCR·´Ó¦Ìåϵ25¦ÌL,¸÷·´Ó¦ÎïŨ¶È: 10 ¡Á»º³åÒº2. 5¦ÌL¡¢MgCl2 ( 25 mmol/L)2¦ÌL¡¢4 ÖÖÍÑÑõºËÌǺËËá2 ¦ÌL¡¢Taq ( 5 U /¦ÌL )0. 4¦ÌL, 3 ¶ÔÒýÎï¸÷1. 25 ¦ÌL¡¢Äæ×ªÂ¼Éú³ÉµÄcDNA 4 ¦ÌL¡¢¼ÓË®ÖÁ25 ¦ÌL¡£·´Ó¦³ÌÐò:
5. ²¡¶¾°ëÊý¸ÐȾÁ¿( TCID50)²â¶¨¡¡ÓÃA /½ËÕ±±ÌÁ/122 /2008 (H1 ) ¡¢A /½ËÕ±±ÌÁ/149 /2008(H3) ¡¢B /½ËÕ±±ÌÁ/137 /2008ÒÑÖªÑôÐԱ걾½øÐÐ10- 1 ¡«10- 7Ï¡ÊÍ,ÔÚÒѳ¤³Éµ¥²ãϸ°ûµÄ96¿×΢Á¿Ï¸°û°åÉÏÓÃMDCK²¡±ä·¨½øÐÐTC ID50²â¶¨¡£¸÷Ï¡ÊͶȽÓÖÖ10¿×,ÿ¿×½ÓÖÖ0. 1 mL,½ÓÖÖ·½·¨Í¬²¡¶¾·ÖÀë, 33¡«
6. mRT2PCR¡¢½ºÌå½ð·¨Ãô¸ÐÐÔÊÔÑé¡¡²ÉÓÃmRT2PCR¡¢½ºÌå½ð·¨·Ö±ð¶ÔÉÏÊö¸÷ÐͲ¡¶¾105¡¢104¡¢103¡¢102¡¢101 TCID50 /0. 1 mL½øÐÐ3´ÎÃô¸ÐÐÔ¼ì²âʵÑé¡£
Èý¡¢Í³¼ÆÑ§·½·¨
ʹÓÃSPSS 11. 0 ͳ¼ÆÈí¼þ½øÐзÖÎö,×é¼ä±È½Ï²ÉÓæÖ2 ¼ìÑé¡£
½á¡¡¡¡¹û
Ò»¡¢mRT2PCRÓ뽺Ìå½ð·¨¼ì²â½á¹û
mRT2PCRºÍ½ºÌå½ð·¨¼ì²â¾´«Í³Ï¸°ûÅàÑø¡¢HI¼ø¶¨ÒÑÖªAÐÍÁ÷¸Ð²¡¶¾ÑôÐÔµÄ2008Äê±ÇÑʱ걾58·Ý,ÒÑÖªB ÐÍÁ÷¸Ð²¡¶¾ÑôÐÔ±ÇÑÊÊñ걾20·Ý;ÒÑÖªÁ÷¸Ð²¡¶¾ÒõÐÔ±ÇÑÊÊñ걾40·Ý¡£¼û±í1¡£mRT2PCR¶ÔAÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪÊÇ44.8% (26 /58) ,ÌØÒìÐÔΪ97. 5% ( 39 /40) ;¶ÔBÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ20. 0% (4 /20) ,ÌØÒìÐÔΪ100. 0% (40 /40) ¡£½ºÌå½ð·¨¶ÔAÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ44. 8% ( 26 /58 ) ,ÌØÒìÐÔΪ100. 0% (0 /40) ;¶ÔB ÐÍÁ÷¸Ð²¡¶¾¼ì²âÃô¸ÐÐÔΪ5. 0% ( 1 /20) ,ÌØÒìÐÔΪ97. 5% ( 39 /40) ¡£mRT2PCRºÍ½ºÌå½ð·¨¶ÔA¡¢B ÐÍÁ÷¸Ð²¡¶¾ÑôÐÔ¼ì³öÂʲîÒìÎÞͳ¼ÆÑ§ÒâÒå( P = 0. 872 5¡¢0. 679 4) ¡£¶Ô¾MDCKϸ°û·ÖÀëÅàÑøÑôÐÔµÄ4 ·ÝA /H1¡¢4 ·ÝA /H3ºÍ3·ÝB ÐÍÁ÷¸ÐÅàÑøÒº,mRT2PCRºÍ½ºÌå½ð·¨¼ì²âµÄÑôÐÔ·ûºÏÂÊ¡¢Ðͱð·ûºÏÂʾùΪ100. 0% mRT2PCRÑÇÐÍ·ûºÏÂÊΪ100. 0%¡£¼ûͼ1¡£
¡¡¡¡×¢:MΪMarker; 1ΪA /½ËÕ±±ÌÁ/110 /2007 (H1) ¡¢2ΪA /½ËÕ±±ÌÁ/112 /2007 (H1 ) ¡¢3 ΪA /½ËÕ±±ÌÁ/122 /2008 (H1 ) ¡¢4 ΪA /½ËÕ±±ÌÁ/1209 /2008 ( H1 ) ; 5 ΪA /½ËÕ±±ÌÁ/1266 /2007(H3) ¡¢6ΪA /½ËÕ±±ÌÁ/1272 /2007 (H3) ¡¢7 ΪA /½Ëճ簲/26 /2008 (H3) ¡¢8ΪA /½ËÕ±±ÌÁ/1160 /2008 (H3) ; 9ΪB /½ËÕÄϳ¤/1351 /2007¡¢10ΪB /½ËÕÄϳ¤/126 /2008¡¢11ΪB /½ËÕÄϳ¤/199 /2008; 12ΪH1¡¢H3¡¢BÐÍÁ÷¸Ð²¡¶¾ÑôÐԱ걾mRT2PCR¶ÔÕÕ; 13ΪH1 +H3ÒýÎï¶ÔÒÑÖªB ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ¡¢14 ΪH1 + B ÒýÎï¶ÔÒÑÖªH3ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ¡¢15ΪH3 +BÒýÎï¶ÔÒÑÖªH1ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ
ͼ1¡¡²¡¶¾ÅàÑøÑôÐÔϸ°ûÅàÑøÒºmRT2PCR¼ì²â½á¹û
¶þ¡¢mRT2PCR¡¢½ºÌå½ð·¨¼ì²âÃô¸ÐÐÔÊÔÑé½á¹û
¶ÔÒÑÏ¡ÊͳÉ101 ¡«105 TC ID50 /0. 1 mL µÄÒÑÖªA /½ËÕ±±ÌÁ/122 /2008 (H1 ) ¡¢A /½ËÕ±±ÌÁ/149 /2008 (H3) ¡¢B /½ËÕ±±ÌÁ/137 /2008Á÷¸Ð²¡¶¾·ÖÀëÑôÐԱ걾½øÐÐmRT2PCR¡¢½ºÌå½ð·¨¼ì²âÃô¸ÐÐÔÊÔÑé¡£½á¹ûmRT2PCRµÄÃô¸ÐÐÔΪ103 TC ID50 /0. 1 mL, ¼ûͼ2¡£½ºÌå½ð·¨Ò²Îª103 TC ID50 /0. 1 mL,ÇÒ3´Î½á¹ûÏàͬ¡£¡¡×¢: MΪMarker; 1Ϊ105 TCID50 /0. 1 mL H1¡¢H3¡¢B; 2Ϊ104TCID50 /0. 1 mL H1¡¢H3¡¢B; 3Ϊ103 TCID50 /0. 1 mL H1¡¢H3¡¢B; 4Ϊ102 TCID50 /0. 1 mL H1¡¢H3¡¢B; 5 Ϊ101 TCID50 /0. 1 mL H1¡¢H3¡¢B; 6ΪÑôÐÔ¶ÔÕÕ; 7 ΪH1 + H3 ÒýÎï¶ÔÒÑÖªB ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ; 8 ΪH1 +B ÒýÎï¶ÔÒÑÖªH3ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ; 9 ΪH3 +BÒýÎï¶ÔÒÑÖªH1ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ
ͼ2¡¡mRT2PCRµÄÃô¸ÐÐÔ½á¹û
ÌÖ¡¡¡¡ÂÛ
ÒÔ´«Í³Á÷¸Ð²¡¶¾·ÖÀëÅàÑøºÍH I¼ø¶¨·½·¨Îª±ê×¼,mRT2PCRºÍ½ºÌå½ð·¨¶Ô¾MDCKϸ°ûÅàÑøµÄÑôÐÔÅàÑøÒº½øÐмì²â, 2ÖÖ·½·¨µÄÑôÐÔ¼ì³öÂʺÍA¡¢BÐÍ·ûºÏÂʾùΪ100. 0%¡£mRT2PCR H1 +H3ÒýÎï¶ÔÒÑÖªB ÐÍÁ÷¸Ð²¡¶¾¼ì²â¡¢H1 +B ÒýÎï¶ÔÒÑÖªH3ÐÍÁ÷¸Ð²¡¶¾¼ì²â¡¢H3 +BÒýÎï¶ÔÒÑÖªH1ÐÍÁ÷¸Ð²¡¶¾¼ì²â¶ÔÕÕ¾ùΪÒõÐÔ,˵Ã÷±¾ÊµÑéËùÓõÄÁ÷¸Ð²¡¶¾H1¡¢H3ºÍBÐÍmRT2PCRÒýÎï¾ßÓкܺõÄÌØÒìÐÔ¡£¶Ô¾´«Í³Á÷¸Ð²¡¶¾·ÖÀëÅàÑøºÍH I¼ø¶¨ÎªAÐÍÁ÷¸Ð²¡¶¾58·ÝÑÊÊñ걾¡¢B ÐÍÁ÷¸Ð²¡¶¾20·ÝÑÊÊñ걾,mRT2PCR¶ÔAÐÍÁ÷¸Ð²¡¶¾µÄ¼ì²âÃô¸ÐÐÔΪ44. 8% ,¶ÔB ÐÍÁ÷¸Ð²¡¶¾µÄ¼ì²âÃô¸ÐÐÔΪ20. 0%¡£Õâ¿ÉÄÜÓ벿·Ö±ê±¾ÔÚ-
±¾Ñо¿½á¹ûÏÔʾ,mRT2PCRºÍ½ºÌå½ð·¨µÄ²¡¶¾¼ì³öÃô¸ÐÐÔ¾ùΪ103 TCID50 /0. 1 mL, ÓëVanEldenµÈ[ 5 ]±¨µÀµÄ½á¹û»ù±¾Ïàͬ¡£¶øÔÚ¶ÔÒÉËÆÁ÷¸Ð»¼Õß±ÇÑÊÊÃ×ӱ걾½øÐв¡¶¾·ÖÀëʱ·¢ÏÖ, Ô¼90%×óÓҵı걾ÔÚ½ÓÖÖϸ°ûºó3¡«4 d³öÏÖϸ°û²¡±ä,˵Ã÷´ó²¿·Ö±ÇÑÊÊÃ×ӱ걾Öв¡¶¾º¬Á¿ÔÚ101 ¡«102 TCID50 /0. 1 mL×óÓÒ[ 6 ] ¡£ÓÃPCR¶ÔcDNA½øÐмì²âµÄÃô¸ÐÐÔ¿É´ï0. 02¡«0. 1 TCID50 /0. 1 mL[7 ] ,Æä¼ì²âµÄÃô¸ÐÐÔ²»ÈÝÖÃÒÉ,µ«ÓÉÓÚÁ÷¸Ð²¡¶¾ÎªRN²¡¶¾,»¹Éæ¼°²¡¶¾RNA³éÌá¹ý³ÌÖеÄËðʧÒÔ¼°Èç¹û±ê±¾²»ÊÇÁ¢¼´½øÐÐÄæ×ªÂ¼ÖÁcDNA,¿ÉÄÜ´æÔÚ²¡¶¾RNA½µ½âµÈÎÊÌâ,Òò´ËÎÒÃÇ×¼±¸½øÒ»²½¶Ô±ÇÑÊÊñ걾¾MDCKϸ°ûÅàÑø8¡«18 hºó²ÉÓÃmRT2PCR¼ø¶¨ºÍÆäËû·½·¨µÄÑо¿,¿Ë·þ´«Í³ÅàÑø·ÖÀëºÍH I¼ø¶¨ÐèҪʱ¼ä½Ï³¤¡¢ÐèÒªÁ÷¸Ð²¡¶¾ÐÍÌØÒìÐÔ¿¹ÑªÇåºÍPCR·´Ó¦ÌåϵËùÐèÒªµÄÓÐЧģ°åÊýÎÊÌâ,ÒÔÌá¸ß¼ì²âµÄÃô¸ÐÐÔ¡£
²Î¿¼ÎÄÏ×
[ 1 ] ¡¡¹ùÔª¼ª,³ÌСö©. Á÷ÐÐÐÔ¸Ð𲡶¾¼°ÆäʵÑé¼¼Êõ[M ]. ±±¾©:ÖйúÈýÏ¿³ö°æÉç, 1997: 138.
[ 2 ] ¡¡Bucher DJ,Mikhail A, Popp le S, et al. Rap id detectionof type A influenza viruseswith monoclonal antibodies tothe M p rotein (Ml) by enzyme2linked immunosorbent assay and time2resolved fluoroimmunoassay[ J ]. J ClinMicrobio, 1991, 11 (5) : 248422488.
[ 3 ] ¡¡Park SY, Shimizu H, Adachi S, et al.
[ 4 ] ¡¡Yamada A, Lam L, Tam JS. Typ ing and subtyp ing ofinfluenza viruses and resp iratory syncytial viruses bymultip lex RT2PCR [ J ]. Int Congr Ser, 2004, 12(63) : 3812385.
[ 5 ] ¡¡Van Elden LJ,NijhuisM, Schipper P, et al. Simultaneousdetection of influenza viruses A and B using real2timequantitative PCR[ J ]. J ClinMicrobiol, 2001, 1 (39) :1962200.
[ 6 ] ¡¡¹¢¹áÒ». Á÷Ðв¡Ñ§[M ]. ±±¾©:ÈËÃñÎÀÉú³ö°æÉç,1984: 35240.
[ 7 ] ¡¡Ò¦¡¡èò, ÕŲÊÔÆ, ³ÂÖÇΰ,µÈ. Ó¦ÓÃPCR 2RFLP¼¼Êõ½øÐÐÒÒÐÍÁ÷¸Ð²¡¶¾¼ø±ðµÄʵÑéÑо¿[ J ]. ÖйúÎÀÉú¼ìÑéÔÓÖ¾, 2007, 17 (11) : 194821951.