µ—«Õ¬à“ ß

Application of Multiplex RT-PCR for Diagnosis of Influenza
¡“ Ÿà§π ®π°√–∑—Ëߪï æ.». 2540 æ∫ºŸâªÉ«¬µ‘¥‡™◊ÈÕ
‰¢âÀ«—¥π° A/H5N1 ®“° —µ«åªï°‡ªìπ§√—Èß·√°∑’Ë
ŒàÕß°ß ¡’ºŸâªÉ«¬ 18 √“¬ ·≈–‡ ’¬™’«‘µ 6 √“¬ ´÷Ëß
π—∫«à“¡’Õ—µ√“ªÉ«¬µ“¬ Ÿß ∑—Ë«‚≈°æ“°—πÀ«—Ëπ«‘µ°«à“
„πÕ𓧵®–‡°‘ ¥ °“√√–∫“¥„À≠à ¢ Õ߇™◊È Õ ‰¢â À «— ¥
„À≠à “¬æ—π∏ÿå„À¡à Õߧ尓√Õπ“¡—¬‚≈°‰¥â«“ß·ºπ
‡ΩÑ “ √–«— ß ‚√§‰¢â À «— ¥ „À≠à ∑—È ß „π§π·≈– — µ «å Õ ¬à “ ß
‡¢â¡·¢Áß ®“°π—Èπ®÷ßæ∫°“√µ‘¥‡™◊ÈÕ‰¢âÀ«—¥π°®“°
—µ«åªï°¡“ Ÿà§πÕ’°‡ªìπ√–¬– ªï æ.». 2542 ∑’ËŒàÕß°ß
æ∫°“√µ‘¥‡™◊ÕÈ ‰¢âÀ«—¥„À≠à A/H9N2 ®“°‰°à¡“ Ÿ§à π
¡’ºŸâªÉ«¬ 2 √“¬ ·µà¡’Õ“°“√‰¡à√ÿπ·√ß ªï æ.». 2546
∑’˪√–‡∑»‡π‡∏Õ√å·≈π¥å ¡’√“¬ß“πºŸâªÉ«¬ 83 √“¬
µ‘ ¥ ‡™◊È Õ ®“° — µ «å ªï ° „πø“√å ¡ ∑”„Àâ — µ «·æ∑¬å
‡ ’¬™’«‘µ 1 √“¬ º≈°“√·¬°‡™◊ÈÕæ∫‡ªì𠓬æ—π∏ÿå
A/H7N7 ·≈–„πªï≈à“ ÿ¥ æ.». 2547 - 2548 ¡’
À≈“¬ª√–‡∑»„π·∂∫‡Õ‡™’¬ ∑’˪√– ∫«‘°ƒµ°“√≥å
¢Õß‚√§‰¢âÀ«—¥π°√–∫“¥„π —µ«åªï°Õ¬à“ß√ÿπ·√ß ¡’
—µ«åªï°≈⡵“¬‡ªìπ®”π«π¡“° √«¡∑—Èßæ∫ºŸâªÉ«¬µ‘¥
‡™◊ÈÕ‰¢âÀ«—¥π°„πª√–‡∑»‡«’¬¥π“¡ °—¡æŸ™“ ·≈–
„πª√–‡∑»‰∑¬æ∫°“√√–∫“¥¢Õ߇™◊ÈÕ Ÿà§πµ—Èß·µà
‡¥◊Õπ¡°√“§¡ æ.». 2547 ·≈–æ∫ºŸâªÉ«¬‡æ‘Ë¡¢÷Èπ
‡√◊ËÕ¬Ê ®π∂÷߇¥◊Õπµÿ≈“§¡ æ.». 2547 √«¡∑—Èß ‘Èπ
17 √“¬ ‡ ’¬™’«‘µ 12 √“¬ §‘¥‡ªìπÕ—µ√“ªÉ«¬µ“¬
√âÕ¬≈– 70.59 ´÷Ëßπ—∫«à“ Ÿß¡“° à«π‰¢âÀ«—¥„À≠à
„π§πæ∫°“√√–∫“¥¬àÕ¬‰¥â∑ÿ°ªï ·≈–æ∫ºŸâªÉ«¬
Ÿß ÿ¥„π™à«ßƒ¥ŸΩ𠵓¡√“¬ß“π¢Õß ”π—°√–∫“¥
°√–∑√«ß “∏“√≥ ÿ¢ æ∫ºŸâªÉ«¬‰¢âÀ«—¥„À≠à‡©≈’ˬ
ªï≈– 40,000 √“¬(2, 3)
«‘ ∏’ ° “√µ√«®«‘ π‘ ® ©— ¬ ‰«√— ‰¢â À «— ¥ „À≠à ∑’Ë
Õߧ尓√Õπ“¡—¬‚≈°·π–π” ”À√—∫ÀâÕߪؑ∫—µ‘°“√
„π‡§√◊Õ¢à“¬ §◊Õ °“√·¬°‡™◊ÈÕ ´÷Ëß “¡“√∂·¬°‡™◊ÈÕ
∑”‰¥â∑ß—È „π‰¢à‰°àø°í À√◊Õ„π‡´≈≈å‡æ“–‡≈’¬È ß ·≈–µ√«®
æ‘ Ÿ®π凙◊Èե⫬«‘∏’ Indirect immunofluorescent
assay (IFA) ®“°π—Èπ®÷ß®”·π°∑—¬ªá·≈– —∫∑—¬ªá
38
Malinee Chittaganpitch et al.
¥â«¬«‘∏’ Hemagglutination inhibition (HI)(4)
´÷ËßµâÕß„™â‡«≈“Õ¬à“ßπâÕ¬ 1 - 2 —ª¥“Àå ‡™◊ÈÕ∑’Ë
·¬°‰¥â„πÀâÕߪؑ∫—µ‘°“√‡§√◊Õ¢à“¬®–∂Ÿ°π” à߉ª¬—ß
Õߧ尓√Õπ“¡—¬‚≈°‡æ◊ËÕ«‘‡§√“–Àå§ÿ≥≈—°…≥–∑“ß
·Õ𵑇®π·≈–®”·π°∂÷ß “¬æ—π∏ÿå ´÷ËߢâÕ¡Ÿ≈∑’ˉ¥â¡’
§«“¡ ”§—≠¡“°µàÕ°“√§—¥‡≈◊Õ° “¬æ—π∏ÿå«—§´’π∑’Ë¡’
ª√– ‘∑∏‘¿“æ¡“„™â„π·µà≈–ªï ·¡â«à“«‘∏’°“√·¬°‡™◊ÈÕ
®–‡ªìπª√–‚¬™πåµàÕ°“√§—¥‡≈◊Õ° “¬æ—π∏ÿå‡æ◊ËÕº≈‘µ
«—§´’π¡“„™â„π°“√§«∫§ÿ¡·≈–ªÑÕß°—π‚√§ ·µà„π
°√≥’∑’Ë¡’°“√√–∫“¥¢Õß‚√§µâÕß°“√º≈°“√µ√«®
¬◊π¬—π∑“ßÀâÕߪؑ∫—µ‘°“√∑’Ë√«¥‡√Á« ´÷Ëß«‘∏’°“√·¬°
‡™◊ÈÕ‰¡à “¡“√∂√“¬ß“πº≈‰¥â∑—πµàÕ‡Àµÿ°“√≥å Õ’°
∑—Èß¡’¢âÕ®”°—¥„π°“√‡°Á∫·≈–π” àßµ—«Õ¬à“ß∑’˵âÕß„Àâ
§«“¡√–¡—¥√–«—߇ªìπ摇»…‡π◊ÕË ß®“°‡™◊ÕÈ ‰«√— µ“¬ßà“¬
‡ªìπº≈°√–∑∫µàÕ°“√‡æ“–·¬°‡™◊ÈÕ ¥—ßπ—Èπ °“√
æ—≤π“«‘∏’µ√«®«‘π‘®©—¬„À≥âº≈∑’Ë√«¥‡√Á« ®÷߇ªìπ
ª√–‚¬™πåÕ¬à“߬‘ËßµàÕ°“√√—°…“ ªÑÕß°—π·≈–§«∫§ÿ¡
‚√§ ªí®®ÿ∫—π‰¥â¡’°“√æ—≤π“«‘∏’ polymerase chain
reaction (PCR) ‡æ◊ÕË „™â„πß“πµ√«®«‘π®‘ ©—¬‚√§µà“ßÊ
Õ¬à“ß°«â“ߢ«“ß √«¡∑—Èß°“√µ√«®«‘π‘®©—¬‚√§‰¢âÀ«—¥
„À≠à∑’Ë¡’§«“¡‰« Ÿß(5, 6, 7) ·≈–„Àâº≈‡√Á«°«à“«‘∏’°“√
·¬°‡™◊ÈÕ πÕ°®“°π’È º≈º≈‘µ¢Õß«‘∏’ PCR ¬—ß
“¡“√∂π”¡“«‘‡§√“–ÀåÀ“‚§√ß √â“ß√–¥—∫‚¡‡≈°ÿ≈
¢Õßµ—«‡™◊ÈÕ ∑”„Àâπ—°«‘∑¬“»“ µ√å “¡“√∂‡¢â“„®
∂÷ß°≈‰°·≈–Àπâ“∑’Ë¢Õß‚ª√µ’π∑’Ë¡’∫∑∫“∑„π°“√
°àÕ‚√§Õ’°¥â«¬
„π°“√»÷°…“§√—Èßπ’ȉ¥âπ”‡∑§π‘§ multiplex
RT-PCR ¡“„™â„π°“√µ√«®«‘π‘®©—¬‚√§‰¢âÀ«—¥„À≠à
‡ª√’¬∫‡∑’¬∫°—∫«‘∏’°“√·¬°‡™◊ÈÕ„π‡´≈≈å‡æ“–‡≈’Ȭß
´÷ßË ‡ªìπ«‘∏¡’ “µ√∞“πµ“¡∑’ÕË ß§å°“√Õπ“¡—¬‚≈°·π–π”
‚¥¬°“√∑¥ Õ∫§«“¡∂Ÿ ° µâ Õ ß¢Õß«‘ ∏’ «‘ ‡ §√“–Àå
(method validation) ∑”„À⇰‘¥§«“¡¡—Ëπ„®„π
°“√ªØ‘∫—µ‘ß“π¢Õ߇®â“Àπâ“∑’ËÀâÕߪؑ∫—µ‘°“√¥â«¬
°“√ª√–¬ÿ°µå„™â«‘∏’ Multiplex RT-PCR „π°“√µ√«®«‘π‘®©—¬
«— ¥ÿ·≈–«‘∏’°“√
‡°Á∫µ—«Õ¬à“ß throat swab ®“°ºŸâªÉ«¬ ARI
(acute respiratory infection) ∑’Ë¡“√—∫°“√√—°…“ ≥
»Ÿπ¬å∫√‘°“√ “∏“√≥ ÿ¢ 17 °√ÿ߇∑æ¡À“π§√ µ—Èß·µà
‡¥◊Õπ¡°√“§¡ æ.». 2546 ∂÷߇¥◊Õπ¡‘∂ÿπ“¬π æ.».
2546 ®”π«π 110 √“¬ ‚¥¬„ à throat swab ·™à„π
À≈Õ¥∑’Ë¡’πÈ”¬“ viral transport media (VTM)
ª√‘¡“≥ 2 ¡‘≈≈‘≈‘µ√µàÕÀ≈Õ¥ „ à°√–µ‘°πÈ”·¢Áß
π” àßÀâÕߪؑ∫—µ‘°“√ À“°‰¡à “¡“√∂π” à߉¥â∑—π∑’
„Àâ π”‡¢â “ µŸâ ‡ ¬Á π 4 Õß»“‡´≈‡´’ ¬ ·≈–π” à ß
ÀâÕߪؑ∫—µ‘°“√¿“¬„π 48 ™—Ë«‚¡ß ‚¥¬·™à„π°√–µ‘°
æ√âÕ¡πÈ”·¢Áß
°“√‡µ√’¬¡µ—«Õ¬à“ß
π”À≈Õ¥ throat swab ‡¢¬à“¥â«¬‡§√◊ÕË ß Vortex
π“π 15 «‘π“∑’ „™â transfer pipette ¥Ÿ¥µ—«Õ¬à“ߢ÷Èπ
≈ߪ√–¡“≥ 5 - 10 «‘π“∑’ ·∫àßµ—«Õ¬à“ßÕÕ°‡ªìπ
2 à«π à«π∑’Ë 1 ”À√—∫°“√·¬°‡™◊ÈÕª√‘¡“≥ 1
¡‘≈≈‘≈‘µ√ ‡°Á∫∑’Ë 4 Õß»“‡´≈‡´’¬ ‡æ◊ËÕ∑”°“√·¬°
‡™◊ÈÕ¿“¬„π«—π‡¥’¬«°—π à«π∑’Ë 2 ”À√—∫ °—¥ RNA
ª√‘¡“≥ 1 ¡‘≈≈‘≈‘µ√ ‡°Á∫∑’Ë -20 Õß»“‡´≈‡´’¬ ‡æ◊ËÕ√Õ°“√∑¥ Õ∫¥â«¬«‘∏’ multiplex RT-PCR «‘∏’
µ√«®«‘π‘®©—¬ ·∫àßÕÕ°‡ªìπ 2 «‘∏’
1. °“√·¬°‡™◊ÈÕ π”µ—«Õ¬à“ß∑’ˇµ√’¬¡·≈â«
( à«π∑’Ë 1) ¡“ªíòπ¥â«¬§«“¡‡√Á« 5,000 √Õ∫/π“∑’
π“π 15 π“∑’ ¥Ÿ¥ à«π„ ¡“‡æ“–‡™◊ÈÕ ‚¥¬À¬¥≈ß
„π¢«¥‡≈’Ȭ߇´≈≈å¢π“¥ 25 µ“√“߇´πµ‘‡¡µ√ ∑’Ë¡’
‡´≈≈å Madine Darby Canine Kidney (MDCK) Õ“¬ÿ
24 - 48 ™—Ë«‚¡ß ª√‘¡“≥ 0.5 ¡‘≈≈‘≈‘µ√ µàÕ¢«¥
∫à¡∑’ËÕÿ≥À¿Ÿ¡‘ 36 ± 1 Õß»“‡´≈‡´’¬ π“π 7 - 10
«—π µ√«®¥Ÿ°“√‡ª≈’ˬπ·ª≈ß√Ÿª√à“ߢÕ߇´≈≈å (CPE
= cytopathic effect) ∑ÿ°«—π µ—«Õ¬à“ß∑’Ë∑”„À⇴≈≈å
‡æ“–‡≈’Ȭ߇°‘¥°“√‡ª≈’ˬπ·ª≈ß√Ÿª√à“ߢÕ߇´≈≈å ®–
∂Ÿ°¢Ÿ¥‡´≈≈å¡“ªíòπ≈â“ß ·≈–π”µ–°Õπ¢Õ߇´≈≈å∑’˪íòπ
‰¥â ¡“À¬¥≈ß∫π ‰≈¥åÀ≈ÿ¡ º÷Ë߉«â„Àâ·Àâß ®“°π—Èπ
®÷ßπ” ‰≈¥å¡“·™à„π cold acetone ∑’Ë -20 Õß»“-
¡“≈‘π’ ®‘µµ°“πµåæ‘™¬å ·≈–§≥–
‡´≈‡´’¬ π“π 10 π“∑’ º÷Ëß„Àâ·Àâß ·≈â«À¬¥¥â«¬
·Õπµ‘∫Õ¥’®”‡æ“–µàÕ‡™◊ÈÕ influenza group A ·≈–
influenza group B (‰¥â√—∫°“√ π—∫ πÿπ®“°Õߧ尓√
Õπ“¡—¬‚≈°) À≈ÿ¡≈– 20 ‰¡‚§√≈‘µ√ π” ‰≈¥å«“ß
„π°≈àÕ߇°Á∫§«“¡™◊πÈ ∫à¡„πµŸÕâ ∫Õÿ≥À¿Ÿ¡‘ 37 Õß»“‡´≈‡´’¬ π“π 30 π“∑’ ‡¡◊ËÕ§√∫‡«≈“ π” ‰≈¥å¡“
≈â“ߥ⫬ phosphate buffer saline 3 §√—Èß º÷Ëß„Àâ·Àâß
·≈â«À¬¥¥â«¬ goat anti-mouse IgG conjugate FITC
À≈ÿ¡≈– 20 ‰¡‚§√≈‘µ√ π” ‰≈¥å«“ß„π°≈àÕ߇°Á∫
§«“¡™◊Èπ ∫à¡„πµŸâÕ∫Õÿ≥À¿Ÿ¡‘ 37 Õß»“‡´≈‡´’¬ π“π 30 π“∑’ ‡¡◊ËÕ§√∫‡«≈“ π” ‰≈¥å¡“≈â“ߥ⫬
phosphate buffer saline 4 §√—Èß º÷Ëß„Àâ·Àâß ·≈â«À¬¥
mounting fluid „Àâ∑—Ë«·ºàπ ‰≈¥åªî¥∑—∫¥â«¬ glass
slip 𔉪 àÕߥ⫬°≈âÕßø≈ŸÕÕ‡√ ‡´πµå ‡´≈≈å∑’Ë¡’
°“√µ‘¥‡™◊ÈÕ®–‡ÀÁ𵑥 ’‡¢’¬«‡√◊Õß· ß à«πµ—«Õ¬à“ß
∑’Ë ‰ ¡à ∑”„Àâ ‡ °‘ ¥ °“√‡ª≈’Ë ¬ π·ª≈ß√Ÿ ª √à “ ߢÕ߇´≈≈å
®–π”πÈ”‡≈’¬È ߇™◊ÕÈ §√—ßÈ ·√°¡“‡æ“–‡™◊ÕÈ µàÕÕ’°§√—ßÈ Àπ÷ßË
‡¡◊ËÕ§√∫ 7 «—π ‰¡à«à“‡´≈≈å®–‡°‘¥°“√‡ª≈’ˬπ·ª≈ß
√Ÿª√à“ßÀ√◊Õ‰¡à ®–π”‡´≈≈å¡“¬âÕ¡¥â«¬«‘∏’ Indirect
immunofluorescent assay (IFA) ‡æ◊ËÕ¬◊π¬—πº≈
§√—Èß ÿ¥∑⓬
2. «‘∏’ multiplex RT-PCR
°“√ °—¥ RNA ®“°µ—«Õ¬à“ß π”µ—«Õ¬à“ß
∑’ˇµ√’¬¡·≈â« ( à«π∑’Ë 2) ª√‘¡“≥ 250 ‰¡‚§√≈‘µ√
„ à≈ß„π micro centrifuge tube ¢π“¥ 2 ¡‘≈≈‘≈‘µ√
‡µ‘¡ 1.0 M Dithiotheritol (DTT) 10 ‰¡‚§√≈‘µ√
µàÕÀ≈Õ¥ ‡¢¬à“¥â«¬‡§√◊ËÕß Vortex π“π 15 «‘π“∑’
∫à¡∑’ËÕÿ≥À¿Ÿ¡‘ÀâÕß π“π 10 π“∑’ ‡µ‘¡ lysis buffer
(RAV 1 containing carrier RNA) ¢Õß™ÿ¥πÈ”¬“
°—¥ RNA ª√‘¡“≥ 600 ‰¡‚§√≈‘µ√ À≈—ß®“°π—Èπ
ªØ‘∫µ— µ‘ “¡§Ÿ¡à Õ◊ Viral RNA extraction kit ¢Õß∫√‘…∑—
ºŸºâ ≈‘µ (NucleoSpin RNAVIRUS, MACHEREYNAGEL GmbH&Co, KG, GERMANY)‚¥¬
¢—ÈπµÕπ ÿ¥∑⓬„π°“√∑” elution à«π¢Õß nucleic
acid „Àâ„™â preheated Rnase free water 70 ‰¡‚§√≈‘µ√
∑’ËÕÿ≥À¿Ÿ¡‘ 70 Õß»“‡´≈‡´’¬ RNA ∑’Ë °—¥‰¥â„Àâ
39
Application of Multiplex RT-PCR for Diagnosis of Influenza
‡°Á∫√—°…“∑’ËÕÿ≥À¿Ÿ¡‘ -70 Õß»“‡´≈‡´’¬ ‡æ◊ËÕ
√Õ°“√∑” multiplex RT-PCR
«‘∏’∑” multiplex RT-PCR ∑”°“√∑¥ Õ∫
„π PCR tube ¢π“¥ 0.2 ¡‘≈≈‘≈‘µ√ ‚¥¬¡’§«“¡
‡¢â¡¢âπ¢ÕßÕߧåª√–°Õ∫µà“ßÊ ¥— ß π’È Rnase free
water 10.75 ‰¡‚§√≈‘µ√, 20 unit/ul Rnase inhibitor
0.25 ‰¡‚§√≈‘µ√, 10 mM dNTP mix 1.0 ‰¡‚§√≈‘µ√,
5x buffer 5.0 ‰¡‚§√≈‘µ√, Enzyme mix 1.0
‰¡‚§√≈‘µ√ (One Step RT-PCR Kit, QIAGEN
co., ltd, GERMANY) 25 µ M primer A/NP-F,
A/NP-R(6), B/HA-F, B/HA-R(8) Õ¬à“ß≈– 0.5
‰¡‚§√≈‘µ√ (µ“√“ß∑’Ë 1) ‡µ‘¡ RNA ∑’Ë °—¥®“°
µ—«Õ¬à“ß 5 ‰¡‚§√≈‘µ√ º ¡„À⇢⓰—π‚¥¬„™â PCR
microspin ®“°π—Èππ”‡¢â“‡§√◊ËÕß PCR thermocycler
‚¥¬µ—Èß‚ª√·°√¡¢—ÈπµÕπ¥—ßπ’È ¢—ÈπµÕπ∑’Ë 1 reverse
transcription ∑’ËÕÿ≥À¿Ÿ¡‘ 50 Õß»“‡´≈‡´’¬ π“π
30 π“∑’ ®”π«π 1 √Õ∫ ¢—ÈπµÕπ∑’Ë 2 activate Taq
DNA polymerase ∑’ËÕÿ≥À¿Ÿ¡‘ 95 Õß»“‡´≈‡´’¬ π“π 15 π“∑’ ®”π«π 1 √Õ∫ ¢—πÈ µÕπ∑’Ë 3 denaturation
Malinee Chittaganpitch et al.
∑’ËÕÿ≥À¿Ÿ¡‘ 94 Õß»“‡´≈‡´’¬ π“π 1 π“∑’ µ“¡¥â«¬
annealing ∑’ËÕÿ≥À¿Ÿ¡‘ 55 Õß»“‡´≈‡´’¬ π“π 1 π“∑’
µ“¡¥â«¬ extension ∑’ËÕÿ≥À¿Ÿ¡‘ 72 Õß»“‡´≈‡´’¬ π“π 1 π“∑’ ®”π«π 40 √Õ∫ ¢—ÈπµÕπ∑’Ë 4 final
extension ∑’ÕË ≥
ÿ À¿Ÿ¡‘ 72 Õß»“‡´≈‡´’¬ π“π 7 π“∑’
®”π«π 1 √Õ∫ ¢—ÈπµÕπ∑’Ë 5 Õÿ≥À¿Ÿ¡‘ 4 Õß»“
‡´≈‡´’¬ µ≈Õ¥‡«≈“
°“√µ√«® Õ∫ PCR product π” PCR
product ∑’ˉ¥â¡“µ√«® Õ∫·∂∫¥’‡ÕÁπ‡Õ (band)
‚¥¬°“√∑” gel electrophoresis º ¡ PCR product
10 ‰¡‚§√≈‘µ√ °—∫ loading buffer 1 ‰¡‚§√≈‘µ√
À¬¥≈ß„π√àÕߢÕß 2% agarose gel ∑’Ë¡’§«“¡‡¢â¡¢âπ¢Õß ethidium bromide 0.1 ¡‘≈≈‘°√—¡µàÕ¡‘≈≈‘≈µ‘ √
‚¥¬„™â°√–· ‰øøÑ“ 100 ‚«≈µå π“π 45 π“∑’ ‡¡◊ËÕ
§√∫‡«≈“π” gel ‡¢â“‡§√◊ËÕß UV transiluminator
‡æ◊ÕË ∫—π∑÷°¿“æ π”¿“æ‚æ≈“√Õ¬¥å∑‰’Ë ¥â¡“µ√«® Õ∫
band ¢Õßµ—«Õ¬à“ß°—∫ RNA influenza positive
control ·≈– DNA marker
µ“√“ß∑’Ë 1 · ¥ß™◊ËÕ primer ≈”¥—∫𑫧≈’‚Õ‰∑¥å·≈–®”π«π‡∫ ¢Õß PCR product
Primer name (Ref.)
Sequence (5û --- 3û)
A/NP- F (6)
GCT TCA AAT GAG AAC ATG GA
A/NP- R (6)
B/HA-F54 (8)
TTG TCT CCG AAG AAA TAA GA
CTG CAC TGG (A/G)AT AAC ATC GTC AAA
CTC ACC
B/HA-R874 (8)
TCA CCA ATT AAA GGC AAG GAC CCT
TTT ATT
40
Product size
380
µ√«®À“ nucleoprotein
gene ¢Õß influenza
group A
820
µ√«®À“ hemagglutinin
gene ¢Õß influenza
group B
°“√ª√–¬ÿ°µå„™â«‘∏’ Multiplex RT-PCR „π°“√µ√«®«‘π‘®©—¬
¡“≈‘π’ ®‘µµ°“πµåæ‘™¬å ·≈–§≥–
°“√ª√–‡¡‘πª√– ‘∑∏‘¿“æ¢Õß«‘∏’µ√«®(9)
«‘∏’„À¡à
º≈∫«°
º≈≈∫
«‘∏’¡“µ√∞“π
º≈∫«°
º≈≈∫
True pos. (TP) False pos. (EP). (FN) TP + FP
False neg.(FN) True neg. (TN)
FN + TN
TP + FN
FP + TN
N
= TP Ó 100
TP + FN
§«“¡®”‡æ“– (specificity)
= TN Ó 100
TN + FP
Õ—µ√“º≈∫«°≈«ß (false positive rate) = FP Ó 100
TN + FP
Õ—µ√“º≈≈∫≈«ß (false negative rate) = FN Ó 100
TP + FN
§«“¡∂Ÿ°µâÕߢÕß«‘∏’ (accuracy)
= (TP + TN) Ó 100
N
§«“¡‰« (sensitivity)
º≈
º≈°“√·¬°‡™◊È Õ ‰¢â À «— ¥ „À≠à „ π‡´≈≈å ‡ æ“–
‡≈’È¬ß ´÷Ë߇ªìπ«‘∏’¡“µ√∞“π ®“°®”π«πµ—«Õ¬à“ß throat
swab 110 √“¬ æ∫µ—«Õ¬à“ß∑’Ë∑”„À⇰‘¥°“√‡ª≈’ˬπ
·ª≈ß√Ÿª√à“ߢÕ߇´≈≈å·≈–¬âÕ¡‡´≈≈å¥â«¬«‘∏’ IFA
‚¥¬„™â·Õπµ‘∫Õ¥’®”‡æ“–µàÕ‡™◊ÈÕ influenza group A
µ‘¥ ’‡¢’¬«‡√◊Õß· ß„Àâº≈∫«°µàÕ influenza A
58 √“¬ „™â·Õπµ‘∫Õ¥’®”‡æ“–µàÕ‡™◊ÕÈ influenza group
B µ‘¥ ’‡¢’¬«‡√◊Õß· ß „Àâº≈∫«°µàÕ influenza B
12 √“¬ à«πµ—«Õ¬à“ß∑’‰Ë ¡à∑”„À⇰‘¥°“√‡ª≈’¬Ë π·ª≈ß
√Ÿª√à“ߢÕ߇´≈≈å π”¡“¬âÕ¡¥â«¬«‘∏’ IFA ‚¥¬„™â
·Õπµ‘∫Õ¥’®”‡æ“–µàÕ‡™◊ÈÕ influenza group A ·≈–
B ‰¡àµ‘¥ ’‡¢’¬«‡√◊Õß· ß „Àâº≈≈∫µàÕ influenza
A ·≈– B 40 √“¬ (µ“√“ß∑’Ë 2)
º≈«‘∏’ multiplex RT-PCR æ∫ PCR product
¢Õßµ—«Õ¬à“ß throat swab ∑’Ë· ¥ß·∂∫¥’‡ÕÁπ‡Õ∑’Ë
380 bp µ√ß°—∫·∂∫¥’‡ÕÁπ‡Õ¢Õß positive control
influenza A „Àâº≈ positive band ¢Õß influenza A
62 √“¬ µ—«Õ¬à“ß∑’Ë· ¥ß·∂∫¥’‡ÕÁπ‡Õ∑’Ë 820 bp
µ√ß°—∫·∂∫¥’‡ÕÁπ‡Õ¢Õß positive control influenza
B „Àâº≈ positive band ¢Õß influenza B 10 √“¬
·≈– PCR product ∑’ˉ¡à· ¥ß·∂∫¥’‡ÕÁπ‡Õ„Àâº≈
≈∫µàÕ influenza A ·≈– influenza B 38 √“¬
(µ“√“ß∑’Ë 2 ·≈–¿“æ∑’Ë 1)
º≈°“√ª√–‡¡‘πª√– ‘∑∏‘¿“æ¢Õß«‘∏°’ “√µ√«®
æ∫«à“ º≈°“√µ√«®«‘π‘®©—¬‚√§‰¢âÀ«—¥„À≠à¥â«¬«‘∏’
multiplex RT-PCR ¡’ § «“¡‰«√â Õ ¬≈– 97.14
§«“¡®”‡æ“–√â Õ ¬≈– 90.00 „Àâ º ≈∫«°¢Õß«‘ ∏’
multiplex RT-PCR µ√ß°—∫«‘∏’·¬°‡™◊ÈÕ 68 √“¬
¡’º≈∫«°≈«ß 4 √“¬ (√âÕ¬≈– 10) „Àâº≈≈∫µ√ß°—π
36 √“¬ ¡’º≈≈∫≈«ß 2 √“¬ (√âÕ¬≈– 2.85)
·≈–§«“¡∂Ÿ ° µâ Õ ß¢Õß«‘ ∏’ multiplex RT-PCR
√âÕ¬≈– 94.54 (µ“√“ß∑’Ë 3)
41
Application of Multiplex RT-PCR for Diagnosis of Influenza
Malinee Chittaganpitch et al.
µ“√“ß∑’Ë 2 º≈°“√µ√«®«‘ π‘ ® ©— ¬ ‚√§‰¢â À «— ¥ „À≠à ™ π‘ ¥ ‡Õ ·≈–∫’ √–À«à“ß«‘∏’°“√·¬°‡™◊ÈÕ ·≈–«‘∏’
Multiplex RT-PCR
º≈°“√µ√«®«‘π‘®©—¬
«‘∏’µ√«®
‰¢âÀ«—¥„À≠à™π‘¥∫’
º≈≈∫
√«¡
°“√·¬°‡™◊ÈÕ
58
12
40
110
Multiplex RT-PCR
62
10
38
110
Marker
control influenza A
control influenza B
‰¢âÀ«—¥„À≠à™π‘¥‡Õ
310
281
234
194
▲
1,353
1,078
872
603
820 bp
▲
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
380 bp
¿“æ∑’Ë 1 · ¥ß·∂∫¥’‡ÕÁπ‡Õ¢Õß PCR product ®“°µ—«Õ¬à“ß throat swab 22 µ—«Õ¬à“ß ‚¥¬™àÕß∑’Ë 1 §◊Õ DNA
marker ™àÕß∑’Ë 24 §◊Õ control Influenza A ™àÕß∑’Ë 25 §◊Õ control Influenza B ™àÕߢÕßµ—«Õ¬à“ß∑’Ë·∂∫¥’‡ÕÁπ‡Õ
„Àâº≈∫«° Influenza A (380 bp) §◊Õ ™àÕß∑’Ë 2, 4, 5, 6, 11, 14 ™àÕߢÕßµ—«Õ¬à“ß∑’Ë·∂∫¥’‡ÕÁπ‡Õ„Àâº≈∫«°
Influenza B (820 bp) §◊Õ ™àÕß∑’Ë 10, 20, 22 ™àÕߢÕßµ—«Õ¬à“ß∑’Ë„Àâº≈≈∫ (‰¡à· ¥ß·∂∫¥’‡ÕÁπ‡Õ) §◊Õ
™àÕß∑’Ë 7, 8, 9, 12, 13, 16, 17, 18, 19, 21
42
°“√ª√–¬ÿ°µå„™â«‘∏’ Multiplex RT-PCR „π°“√µ√«®«‘π‘®©—¬
¡“≈‘π’ ®‘µµ°“πµåæ‘™¬å ·≈–§≥–
µ“√“ß∑’Ë 3 ‡ª√’¬∫‡∑’¬∫º≈°“√«‘‡§√“–Àå«‘π‘®©—¬‚√§‰¢âÀ«—¥„À≠à ¥â«¬«‘∏’°“√·¬°‡™◊ÈÕ·≈–«‘∏’ multiplex
RT-PCR
«‘∏°’ “√·¬°‡™◊ÕÈ
√«¡
º≈∫«°
º≈≈∫
º≈∫«°
68
4
72
º≈≈∫
√«¡
2
70
36
40
38
110
«‘∏’ Multiplex RT-PCR
§«“¡‰« (sensitivity)
§«“¡®”‡æ“– (specificity)
Õ—µ√“º≈∫«°≈«ß (false positive rate)
Õ—µ√“º≈≈∫≈«ß (false negative rate)
§«“¡∂Ÿ°µâÕߢÕß«‘∏’ (Accuracy)
=
=
=
=
=
97.14%
90.00%
10.00%
2.85%
94.54%
«‘®“√≥å
«‘∏’ multiplex RT-PCR ∑’˧≥–ºŸâ«‘®—¬‰¥âπ”
¡“ª√–¬ÿ°µå„™âµ√«®À“ “√æ—π∏ÿ°√√¡π’ȉ¥â∑”°“√
∑¥ Õ∫·≈–¥—¥·ª≈ß‚¥¬‡√‘Ë¡®“°¢—ÈπµÕπ°“√ °—¥
µ—«Õ¬à“ß‚¥¬‡æ‘¡Ë ¢—πÈ µÕπ°“√∑” prelysis ´’ßË ‰¥âπ”‡Õ“
dithiotheritol (DTT) ·≈– proteinase K ´÷Ë߇ªìπ
“√®”æ«° denaturing protein „ à≈ß„πµ—«Õ¬à“ß
°àÕπ∑’Ë®–„™â™ÿ¥πÈ”¬“ °—¥ æ∫«à“ DTT ∑’˧«“¡‡¢â¡¢âπ
1.0 M „Àâª√‘¡“≥¢Õß RNA ∑’Ë °—¥‰¥â‡¢â¡¢âπ°«à“
°“√‰¡à„ à DTT ª√–¡“≥ 0.5 ‡∑à“µ—« à«π°“√„ à
proteinase K ‰¡à¡º’ ≈µàÕ°“√ °—¥ RNA(10)
„π¢—ÈπµÕπ°“√∑” PCR ‰¥â§—¥‡≈◊Õ° primer
À≈“¬§Ÿà∑’Ë®”‡æ“–µàÕ¬’π å„π à«πµà“ßÊ ¢Õ߇™◊ÈÕ
‰¢âÀ«—¥„À≠à ‡™àπ hemagglutinin nucleoprotein
·≈– non structural protein ´÷Ëß¡’ºŸâ«‘®—¬À≈“¬§≥–
‰¥â»÷°…“ ‡™àπ K. E. Wright ·≈–§≥–(5) K. Okazaki
·≈–§≥–(6) Dean D. Erdman ·≈–§≥–(7) primer
‡À≈à“π’ȉ¥âπ”¡“∑¥ Õ∫°—∫‡™◊ÈÕ‰¢âÀ«—¥„À≠à∑’Ë·¬°‰¥â
„πª√–‡∑»‰∑¬ ·≈–‡™◊ÈÕ‰«√— √–∫∫∑“߇¥‘πÀ“¬„®
µ—«Õ◊πË Ê ‡™àπ parainfluenza virus type3, respiratory
syncytial virus, adeno virus ·≈– rubella virus
´÷Ëßæ∫«à“ primer µàÕ nucleoprotein ¬’π 凙◊ÈÕ‰¢â
À«—¥„À≠à™π‘¥‡Õ ·≈– primer µàÕ hemagglutinin
¬’π å¢Õ߇™◊ÈÕ‰¢âÀ«—¥„À≠à™π‘¥∫’ ¡’§«“¡®”‡æ“– Ÿß
‰¡à‡°‘¥ cross reaction °—∫‡™◊ÈÕ‰«√— √–∫∫∑“߇¥‘π
À“¬„®µ—«Õ◊ËπÊ ·≈–¡’§«“¡‰«µàÕ‡™◊ÈÕ‰¢âÀ«—¥„À≠à‰¥â
¥’°«à“ primer §ŸàÕ◊ËπÊ ·≈– “¡“√∂„™â primer 2 §Ÿàπ’È
√«¡‰«â„πªØ‘°‘√‘¬“À≈Õ¥‡¥’¬«°—π ‚¥¬‰¡à‡°‘¥°“√
·¬àß°—π∑”ªØ‘°‘√‘¬“ PCR
43
Application of Multiplex RT-PCR for Diagnosis of Influenza
®“°°“√¥—¥·ª≈ߢ—ÈπµÕπ°“√ °—¥ RNA
®“°µ—«Õ¬à“ß·≈–°“√ª√—∫ ¿“æ∑’ˇÀ¡“– ¡ √«¡∑—Èß
§«“¡‡¢â¡¢âπ¢Õß primer ∑’Ë„™â„π¢—ÈπµÕπ RT-PCR
‡¡◊ËÕπ”¡“∑¥ Õ∫°—∫µ—«Õ¬à“ß throat swab ®”π«π
110 √“¬ æ∫«à“ «‘∏’ multiplex RT-PCR „Àâº≈∑’Ë
„°≈â ‡ §’ ¬ ß°— ∫ «‘ ∏’ · ¬°‡™◊È Õ ´÷Ë ß ‡ªì π «‘ ∏’ ¡ “µ√∞“π
∑—Èߧ«“¡‰«·≈–§«“¡®”‡æ“–·µà¡’¢âÕ¥’°«à“«‘∏’·¬°
‡™◊ÈÕ∑’Ë„Àâº≈∑’Ë√«¥‡√Á«¿“¬„π 24 ™—Ë«‚¡ß “¡“√∂
µ√«®µ— « Õ¬à “ ߉¥â ®”π«π¡“°„π·µà ≈ –§√—È ß ·≈–
“¡“√∂µ√«®µ—«Õ¬à“ß∑’ˇ™◊ÈÕµ“¬·≈â«´÷Ë߇ªìπ¢âÕ®”°—¥
¢Õß«‘∏’°“√·¬°‡™◊ÈÕ ¥—ßπ—Èπ «‘∏’ multiplex RT-PCR
®÷ß¡’§«“¡‡À¡“– ¡∑’Ë®–π”¡“„™âµ√«®«‘π‘®©—¬‚√§
‰¢â À «— ¥ „À≠à ‡æ◊Ë Õ ª√–‚¬™πå µà Õ °“√ ◊ ∫ «π°“√
√–∫“¥¢Õß‚√§√–∫∫∑“߇¥‘πÀ“¬„®·≈–°“√√—°…“
ºŸâªÉ«¬‚√§‰¢âÀ«—¥„À≠à·≈–‰¢âÀ«—¥π° ‡π◊ËÕß®“°„π
¢≥–π’È¡’¬“∑’Ë„™â√—°…“·≈–ªÑÕß°—π‚√§‰¢âÀ«—¥„À≠à
‡™àπ Oseltamivir À√◊Õ Tamiflu ´÷Ëß¡’§ÿ≥ ¡∫—µ‘
„π°“√¬—∫¬—È߇Õπ‰´¡å neuraminidase ¢Õ߇™◊ÈÕ
‰¢âÀ«—¥„À≠à ¬“π’È®–„Àâº≈¥’„π°“√√—°…“‚¥¬≈¥
§«“¡√ÿπ·√ߢÕß‚√§≈ß·≈–≈¥Õ—µ√“‡ ’ˬ߇ªìπ‚√§
ªÕ¥∫«¡„π°≈ÿà ¡ ºŸâ ªÉ « ¬‰¢â À «— ¥ „À≠à Õ “¬ÿ πâ Õ ¬°«à “
12 ªï ·≈–°≈ÿà¡Õ“¬ÿ¡“°°«à“ 60 ªï‰¥â∂÷ß√âÕ¬≈– 20
·≈– 60 À“°ºŸâªÉ«¬‰¥â√—∫¬“¿“¬„π 2 «—πÀ≈—ß®“°
‡√‘Ë¡¡’Õ“°“√(11) ¥—ßπ—Èπ À“°ºŸâªÉ«¬∑√“∫º≈°“√µ√«®
«‘ π‘ ® ©— ¬ ‰¥â √ «¥‡√Á « ®–‡ªì π ª√–‚¬™πå Õ ¬à “ ߬‘Ë ß µà Õ
°“√√—°…“ºŸâªÉ«¬·≈–≈¥Õ—µ√“°“√ªÉ«¬µ“¬„ÀâπâÕ¬≈ß
√ÿª
«‘∏’ multiplex RT-PCR ‡ªìπ«‘∏’∑’Ë¡’§«“¡‰«
·≈–§«“¡®”‡æ“– Ÿß ‡¡◊ËÕ‡∑’¬∫°—∫«‘∏’°“√·¬°‡™◊ÈÕ∑’Ë
‡ªìπ«‘∏¡’ “µ√∞“π„π°“√µ√«®«‘π®‘ ©—¬‚√§‰¢âÀ«—¥„À≠à
·¡â «à “ «‘ ∏’ ° “√·¬°‡™◊È Õ ¬— ß §ß¡’ § «“¡ ”§— ≠ µà Õ °“√
»÷°…“¥â“π·Õ𵑇®π¢Õ߇™◊ÈÕ·≈–°“√æ—≤π“«—§´’π
·µà°“√π”«‘∏’ multiplex RT-PCR ¡“ª√–¬ÿ°µå„™â
44
Malinee Chittaganpitch et al.
®–™à«¬„Àâ∑√“∫º≈°“√µ√«®∑’Ë√«¥‡√Á« ´÷Ëß®–‡ªìπ
ª√–‚¬™πåµàÕ°“√«“ß·π«∑“ß°“√√—°…“ºŸâªÉ«¬¢Õß
·æ∑¬å ·≈–°“√ ◊∫ «π‚√§‡¡◊ËÕ¡’°“√√–∫“¥
°‘µµ‘°√√¡ª√–°“»
¢Õ¢Õ∫§ÿ≥·æ∑¬å 欓∫“≈ ·≈–‡®â“Àπâ“∑’Ë
»Ÿπ¬å∫√‘°“√ “∏“√≥ ÿ¢ 17 °√ÿ߇∑æ¡À“π§√ ∑’°Ë √ÿ≥“
§—¥‡≈◊Õ°ºŸâªÉ«¬‡æ◊ËÕ∑”°“√‡°Á∫µ—«Õ¬à“ß ·≈–Õߧ尓√
Õπ“¡—¬‚≈°∑’ËÕπÿ‡§√“–ÀåπÈ”¬“‡æ◊ËÕ°“√µ√«®«‘π‘®©—¬
·≈–¢Õ¢Õ∫§ÿ≥ §ÿ≥»‘√‘¡“ ªí∑¡¥‘≈° ∑’Ë°√ÿ≥“„Àâ
§”·π–π”„π°“√‡¢’¬π∫∑§«“¡
‡Õ° “√Õâ“ßÕ‘ß
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Regional Workshop on Human Influenza A
(H5N1), Bangkok: Ministry of Public Health,
19 - 20 February 2004
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& Response (CRS). Cumulative number of
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disease/avian_influenza/en
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°“√ª√–¬ÿ°µå„™â«‘∏’ Multiplex RT-PCR „π°“√µ√«®«‘π‘®©—¬
¡“≈‘π’ ®‘µµ°“πµåæ‘™¬å ·≈–§≥–
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Application of Multiplex RT-PCR for Diagnosis of Influenza
Malinee Chittaganpitch Sunthareeya Waicharoen
and Pranee Thawatsupha
Paiboon Maneewong
National Institute of Health, Department of Medical Sciences, Tiwanond Road, Nonthaburi 11000, Thailand.
ABSTRACT
Diagnosis of Influenza can be performed by several methods. Serological test using pair
sera is one method. It usually gives ambiguous result due to reinfection by viruses of the same group.
Isolation of virus is a sensitive and highly specific method, as well as identification of Influenza virus
strains. However, the method is complicated and takes about 1 - 2 weeks. Multiplex RT-PCR is applied to
detect nucleic acid of Influenza virus as a rapid method that yields a result within 24 hours. Throat swabs
from patients with acute respiratory infection (ARI), at Health Center 17, Bangkok were collected
in this study. Specimens were both cultured in MDCK cell and detected nucleic acid using 2 primers set.
The first primer set, A/NP-F and A/NP-R, is used to detect nucleoprotein gene of influenza A. The second
set, B/HA-F and B/HA-R, is used to detect hemagglutinin gene of influenza B. Evaluation of 110
specimens by multiplex RT-PCR compared with viral isolation as a standard method showed that the
sensitivity, specificity and accuracy of multiplex RT-PCR were 97.14%, 90.00% and 94.54% respectively.
Key words : Influenza, multiplex RT-PCR
45