вټⷽԺо

ʱ䣺2015/2/10 13:07:50

вټⷽԺо

ȷ1 , 2 , žƽ1 , ʩǿ2 , Դ1 , 1 , 1 , 衡ϼ1

(1. мԤ214023; 2. ѧѧԺϺ200032)

  ժҪ:ĿȽ϶ת¼ۺøӦ(mRT2PCR)ͽ𷨼вԺmRT2PCRͽ𷨼⾭ϸͺϸ(H I)ΪA /H1A /H3Bвıñ걾78,Աñ걾40,ϡͳ101 105Ⱦ( TCID50) /0. 1 mL3H1H3BвmRT2PCRAвļΪ44. 8% ,Ϊ97. 5%;BвΪ20. 0% ,Ϊ100. 0%𷨶AвΪ

44. 8% ,Ϊ100. 0%;вΪ5. 0% ,Ϊ97. 5%Ծ20 ڹϸ(MDCK)Ե4A /H14A /H33BҺ,mRT2PCRͽ𷨼ԷͱʾΪ100. 0%mRT2PCRвΪ103 TCID50 /0. 1 mL;𷨼Ϊ103 TCID50 /0. 1 mLۡmRT2PCRͽ𷨵޲; mRT2PCRͽ𷨾Ⱥбñ걾вټ,رǾۼԱл,ʱ걾ҪʵŴ23mRT2PCRɽһȷвH1H3

ؼ:в;;ת¼ۺøӦ;

Research on the specificity and sensitivity of rapid detection methods of influenza virusYOU 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, China; 2. The College of Public Health of FudanUniversity, Shanghai 200032, China)

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. MethodsThe 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. ResultsThe 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 sThere 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


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1. 걾Դ걾ҽԺ

ͯҽԺֲеҽԺڿƹ

мڵҽԺ,Ӿײ(¡38 )ɼ;ֲб,IJɼ걾걾ɼ12 hʵ, 24 hڽвͿټ24 hڲܽʵñ걾70 ±䱣вʵ걾ѡɱҷȷϵA /ձ/122 /2008(H1 ) A /ձ/149 /2008 (H3 ) B /ձ/137 /2008в

2. ԼRNAȡmRT2PCRʹñ

޹˾Takara RNAiso PlusԼ,Ϊ

DV818AƲμ[ 3, 4 ]H1H3B

ֱΪ431210390 bp

ɺݴؼ޹˾ṩ(

ҩ׼S20050106 ) ,ΪFA080301Ϊ

B IO2RAD Mycycler PCR

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1. 20ڹ

ϸ(MDCK)걾Ԥ: 3 mL

150LĿ2ҹѳɵ

MDCKú0. 02 mL /mLĿ

ϴ2,150300 34 

2 h걾ҺάҺ(ŨΪ

5g/mLø50L /mLĿ0. 2%СţѪ׵) ,35 5%CO2, 24 hչ۲ϸ, 1ܺеϸҺ0. 75%ˡOͺϸ΢ѪʵѪ

Һùͳһ·֪ѪH I,вͱ

2. 𷨼⡡Լ˵



3. Takara RNAiso Plus RNAȡ

TakaraԼȡ

4. mRT2PCRTakaraԼ˵mRT2PCRӦϵ25L,ӦŨ: 10 Һ2. 5LMgCl2 ( 25 mmol/L)2LǺLTaq ( 5 U /L )0. 4L, 3 1. 25 Lת¼ɵcDNA 4 Lˮ25 L94 3 min, 94  30 s51  1 min68  1 min,35ѭ68  10 minж: H1 431 bp, H3210 bp,B 390 bp

5. Ⱦ( TCID50)A /ձ/122 /2008 (H1 ) A /ձ/149 /2008(H3) B /ձ/137 /2008֪Ա걾10- 1 10- 7ϡ,ѳɵϸ96΢ϸMDCKTC ID50ⶨϡͶȽ10,ÿ׽0. 1 mL,ַͬ, 3335 5% CO2һжTCID50A /ձ/122 /2008 (H1)Ϊ106. 2 TC ID50 /0. 1 mLA /ձ/149 /2008 ( H3 ) Ϊ106. 5TCID50 /0. 1 mLB /ձ/137 /2008 Ϊ106. 5TCID50 /0. 1 mL

6. mRT2PCR顡mRT2PCR𷨷ֱͲ105104103102101 TCID50 /0. 1 mL3Լʵ

ͳѧ

ʹSPSS 11. 0 ͳз,Ƚϲæ

һmRT2PCR뽺𷨼

mRT2PCRͽ𷨼⾭ͳϸHI֪AвԵ2008ʱ걾58,֪вԱñ걾20;֪вԱñ걾401mRT2PCRAвΪ44.8% (26 /58) ,Ϊ97. 5% ( 39 /40) ;BвΪ20. 0% (4 /20) ,Ϊ100. 0% (40 /40) 𷨶AвΪ44. 8% ( 26 /58 ) ,Ϊ100. 0% (0 /40) ;вΪ5. 0% ( 1 /20) ,Ϊ97. 5% ( 39 /40) mRT2PCRͽ𷨶AвԼʲͳѧ( P = 0. 872 50. 679 4) MDCKϸԵA /H1A /H33Һ,mRT2PCRͽ𷨼ԷͱʾΪ100. 0% mRT2PCRͷΪ100. 0%ͼ1

ע:MΪMarker; 1ΪA /ձ/110 /2007 (H1) 2ΪA /ձ/112 /2007 (H1 ) ΪA /ձ/122 /2008 (H1 ) ΪA /ձ/1209 /2008 ( H1 ) ; 5 ΪA /ձ/1266 /2007(H3) 6ΪA /ձ/1272 /2007 (H3) ΪA /ճ簲/26 /2008 (H3) 8ΪA /ձ/1160 /2008 (H3) ; 9ΪB /ϳ/1351 /200710ΪB /ϳ/126 /200811ΪB /ϳ/199 /2008; 12ΪH1H3BвԱ걾mRT2PCR; 13ΪH1 +H3֪в14 ΪH1 + B ֪H3в15ΪH3 +B֪H1в

ͼ1ϸҺmRT2PCR

mRT2PCR𷨼

ϡͳ101 105 TC ID50 /0. 1 mL ֪A /ձ/122 /2008 (H1 ) A /ձ/149 /2008 (H3) B /ձ/137 /2008вԱ걾mRT2PCR𷨼mRT2PCRΪ103 TC ID50 /0. 1 mL, ͼ2ҲΪ103 TC ID50 /0. 1 mL,3νͬע: MΪMarker; 1Ϊ105 TCID50 /0. 1 mL H1H3B; 2Ϊ104TCID50 /0. 1 mL H1H3B; 3Ϊ103 TCID50 /0. 1 mL H1H3B; 4Ϊ102 TCID50 /0. 1 mL H1H3B; 5 Ϊ101 TCID50 /0. 1 mL H1H3B; 6ΪԶ; 7 ΪH1 + H3 ֪в; 8 ΪH1 +B ֪H3в; 9 ΪH3 +B֪H1в

ͼ2mRT2PCRԽ

֡

ԴͳвH IΪ׼,mRT2PCRͽ𷨶ԾMDCKϸҺм, 2ַԼʺABͷʾΪ100. 0%mRT2PCR H1 +H3֪вH1 +B ֪H3вH3 +B֪H1вվΪ,˵ʵõвH1H3BmRT2PCRкܺõԾͳвH IΪAв58ñ걾в20ñ걾,mRT2PCRAвļΪ44. 8% ,вļΪ20. 0%벿ֱ걾70 䴢һʱԺٽкmRT2PCR й,ñ걾ȡmRT2PCR,Լʻ,Ҫ֪Ⱥۼ, mRT2PCR ǿȡķ,ΪͳIJH IķʱϳȻ𷨳,ֻ20 min,,ֳ,ֻܼA,Ŀǰܽͼ,ûйԼ,Լм۸񰺹,ױԽϵ[ 2 ] mR2PCR 4 h,Ҫһ豸,Ƚ𷨳ɱ,ҿԽͼھۼб,Ҫٳ,ܹṩ͵

оʾ,mRT2PCRͽ𷨵IJԾΪ103 TCID50 /0. 1 mL, VanElden[ 5 ]Ľͬڶл߱ӱ걾вʱԼ90%ҵı걾ڽϸ34 dϸ,˵󲿷ֱӱ걾в101 102 TCID50 /0. 1 mL[ 6 ] PCRcDNAмԿɴ0. 020. 1 TCID50 /0. 1 mL[7 ] ,Բ,вΪRN,RNAеʧԼ걾ת¼cDNA,ܴڲRNA,׼һԱñ걾MDCKϸ818 hmRT2PCRо,˷ͳH IҪʱϳҪвԿѪPCRӦϵҪЧģ,߼

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