%PDF-1.4
%忏嫌
8 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
3 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
11 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
9 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
5 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
10 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
4 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
6 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
1 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
7 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 137>>stream
x???晓жRA-荡@C驒t冀ぐ儋賐哔*}c`;U€盿貨肢誆>a惡u-?痠覫隵?Q滭I|滷鳂忪!\?o椏捬|撂磘bS戀亿?珯瞎榃珇?
endstream
endobj
13 0 obj
<>stream
A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology
10.1128/Spectrum.01267-21
Fei Zhao
Jinxing Lu
Bing Lu
Tian Qin
Xuemei Wang
Xuexin Hou
Fanliang Meng
Xiaona Xu
Tianyi Li
Haijian Zhou
Jianzhong Zhang
Biao Kan
Ying Huang
Zehui Zhang
Di Xiao
Research Article
Research Article
SARS-CoV-2 variants
SNPs
screening and identification
mPCR-MS minisequencing
American Society for Microbiology
Zhao et al.
Copyright 漏 2021 Zhao et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
20211117
2021
ABSTRACT
The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization鈥搕ime of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5鈥壝椻€?03 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection.
IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.
Citation
20210812
20211019
2165-0497
Adobe LiveCycle PDF Generator; modified using iText庐 5.5.13.2 漏2000-2020 iText Group NV (AGPL-version)2021-11-17T22:23:43-08:00
endstream
endobj
14 0 obj
<>stream
x? |
endstream
endobj
15 0 obj
<>stream
x淪??T0T0 B櫆珷慽挴 yw*
endstream
endobj
16 0 obj
<>stream
x? |
endstream
endobj
17 0 obj
<>stream
x淪??T0T0 B櫆珷慽ㄠ挴 yA$
endstream
endobj
18 0 obj
<>stream
x? |
endstream
endobj
19 0 obj
<>stream
x淪??T0T0 B櫆珷慽︵挴 yn)
endstream
endobj
20 0 obj
<>stream
x? |
endstream
endobj
21 0 obj
<>stream
x淪??T0T0 B櫆珷慽∴挴 y€+
endstream
endobj
22 0 obj
<>stream
x? |
endstream
endobj
23 0 obj
<>stream
x淪??T0T0 B櫆珷慽犩挴 y8#
endstream
endobj
24 0 obj
<>stream
x? |
endstream
endobj
25 0 obj
<>stream
x淪??T0T0 B櫆珷慽挴 yS&
endstream
endobj
26 0 obj
<>stream
x? |
endstream
endobj
27 0 obj
<>stream
x淪??T0T0 B櫆珷慽挴 ye(
endstream
endobj
28 0 obj
<>stream
x? |
endstream
endobj
29 0 obj
<>stream
x淪??T0T0 B櫆珷慽む挴 yJ%
endstream
endobj
30 0 obj
<>stream
x? |
endstream
endobj
31 0 obj
<>stream
x淪??T0T0 B櫆珷慽┼挴 y,
endstream
endobj
32 0 obj
<>stream
x? |
endstream
endobj
33 0 obj
<>stream
x淪??T0T0 B櫆珷慽⑧挴 y\'
endstream
endobj
35 0 obj
<>stream
H墝W蛂芨>霱O?? 俤?速-o?帳8暡r燞幠x啘%9?掔M gF枔-U峻E爿O7轾胻鳋鲮輳妩=縘焛肥洰t?跏邳黺{鲱jJ擉z"颟钕掦?蟝?糊 ?╠)鍄柇\氬鐈bW$唀$|枮=讧vlXj?UU飃,Rc;砗睔囻鐅暙楗o芶唆?S?}踉嶜 \9慼湋Nu.痮猥醩"籙獻寶vc[5耊ㄧa?q粝蹮 ^湲孠荠B5ば鋹>u}蛬挻蠌埢鞩?蘆玜嵂攖?-W牥磢四EBVNm==yU=WO^孆饨鐝獯GZ莘m g毆~藜0t灔iqj@] ?歭ǐ箽鎞??'Dl<苣懤M唰v蹘澑`!eG|O肇€MJ誐⒃x_懖L}m_労T氇椷pRぃXg杊_ 箘馳,~訖[鼏搶i蚀4愮'.Q獀U叽郛^1滒?%顭鳊姖N/斩 殜?硞雩??T迎醉xdL巯圿蜈K螡z蒭磣p昝K逳QDc★~tRXi盝ta█(戈嚀e篖鍎4"緻歭鰨 XR棹謜s蹐芓龣垰a嬅4吏橢嗖<;M?鮺Ey甘I昁SRu官=Vw譁Z7脋~摹U楇鲍'%パv?C8I&KY件緷偓邯6輟Av聻{v婥浐托驭S看哮蘃?嗰玊Q眻2E攊w!}<S&挲X踭M兰恺碕e騍递z%Pa郧緛??t*?fg}h7@乪A橆?凾Qw[咃n庿惟顔憇LL麌噕b%g@5媝睑875鑣撐r誱I鋛?jmQ职a顔炈
!y硢嵌nwT桉}譑B潫rl斩迳Evv軼Yㄝ8汰绁鵕wG]=矲2梛QS饶篅#*灎*u粂i跰坈斓隬匢I隠窠略E(毯?啷刌簟瘗J秽?k马偻/閙IM7啪€砋R8wZ|:d?V茜?她x货跻Nu祂%t躬瞟 访q/f瞌晅駭病俪>{曰轜分充儊侰X挲8稈Q=Uu蒗?閴?>\P瑕,|砊9崠-s㈠睥?妀浤#CWa隟呓a縁b>馢x佲?d?€S蒂荏抚[F?民⒓歺莌潸h~!UtqⅫ6音K纠(寕
椤蜈Vm#$诪x襭XEP呬7炙-IyD碆a1DD秹t€(hNヰ$a犷 ?葷l 砣?搫eO3必截c\擟^€o</jR枮n-轌 諢#K洀.n鼬匈朠
擛 TK@!(T}俇N@8鮧劍庿槹論e?Q麸?拢┾&?奀暉諤JM]嘥鏠Jp巫C55僑?_>Rb棝Czd?龜軄y?繿颴鰩c?孓rK栛`x螁Kb臧?Vc?嶜Q既颡(HPT酿?+鎥臝ekI隥囱u寻 壍咢覮T汓汬=F鵇I*蠊拍D{t|y?﹥oK qK翝?x?抖嚓OO?zR騗繉YJ@*}?颜2謌伀=V睭Z,偉SP-?8[点|踺x`4翞衠t)e,篰]?#曀U,y煔<鰜﹠疊蹥EshCx伸瀈嗺#R胮j`禃桸*滸2sV枼?脍圼k霩Sf0加€3樐J<2嬆?h}幧&琄|>H銦厡菩?F礦瀪沲.谲否1孖?絛菽U-!渺慘揠芭?紟﨔憠傟N[昻铎镱l遏滿捑?棖拤vhb笋K€昳&鉳62椧2BT裢i???HYK媜??:项Aza谩髍c浸N塯9ZD5瀡?f2潲>痣5勖0
棽}蚆橏
鹐帔LZp=#洿H蠺蓨藉'陎.= 秀M⒄E扺扸災a呍嬩盨6J?嬪P帤?辷鬋c>歲譋果龐 w鲷?ce羉Z??持\爗?锡3魽壚贜Fr椛(i力c4#n乓k躳IKl罯ヨ军埨夃槜/Q6}繣粜&燑倾??规.?c岋B呥壝悯x枛‘敗.恪庍硺2{
颔蘍睂B菙闆儴屄唪タ{( 腙餔侶^p鼈硼mx瘱A裍{,??缶@tk卢橕w@{?<]a满?駎rV孿婌0洊JE?裷yE踱?緺€甠/#()踳|yg傕x傃Ex坐Z?G?? 希7哴z€d:鴳>岎9.鸭S文脇?謴叁雏ys?瞒蹇湕輓?O酜`jg虹饕慶纮?d%W埂缺?ER鈖m鶤蚣镾Иg殥嶌 瓩檫觋S吖蒣ē,?旧綖YAqf)紾/0+)Z)?y冫蘕_?樛愺pE屷?Z鵷韍奴`?pG?u鬵?*酬 偺煦鞳n洤溙饤焆芳1?X砺4+ 臁h$敲
l婷^r废J亷BJ盘I
H?磠U?Q4}:q]gH?隐C6鱒匁=蒼o1喁a(w馧M嵒颃烙\g瑸&砂軗曟v犬0哐4崵?R紷ㄙ}" ︰R
D2n?树 '笁絨_x綽哅os閧R編隚?檉活'$洵 4牦&;踜餜?脉OG8?*"駬64墽c斖QiM?8[3鵍菇閒?搐w厁a4([?肅玌o/?^藊蒑墼m揞羶*禁ゆ偑?曻夙醠铠EkE&慳臨~59眉椫輯p?v衭痀 6睂o
虉冀毫!崭 f7 I?矤台;h歀锋6Nh貸省食兡%,D3貵鏧k輟nnKS≧(瑫被? m?3瞕0WEC?2_倔=?襄獤R繁 幛6鋖V珡轰6Z齬攰锋碅/?T絒x竨彟!'潒扣2f祐曱[吳VjJ?實腤r剝 ?k?o魑sZ|剜鐊?ズ?>臶s饤w酓芣糷iFv8嚜L
罛龎/},餓Y讞8
)孳k姕╥brE?XRn摅i&0~藌玊羨鑵m尺gL nG*V?鸥酇抒#A┓U+笔/HR轟s皳苔聢蒇@^秧0JHe琩3qs嫊6照萸矛軭pF1dD卑q G秛蛨揆T~罫>d?€?蹡?]槚?濏2L?诮t绝a濓縜]a慽針旂G蟌5' 柤:>辘耰-谊gl爫/鹔up%洺b乖R逆w吊?适zb榗?蔯C畖~檾v臊"2覨+Q;溴~DA烾R槦X":磎爀覲纥蟵d+J=洅墖f雹哉f'抁sq哖邯m?廥揝&瞫{m昛犥Se?宻
黪K济aC~D ?l蟘l貕0蝡蟽4啅烪n?Cc86槔z>萏7頬縒じ1Dvzzb讫?L#獵丘峹u綉j?冏z+ 馸e軪b@蝧鷘FS隷x
孊;犞欹@釶錫SW历&|?槓潉Xku0?幠G E皫眘?齪掝R?朶翨鴜贪~e多, 譧(乮G<檖捔識&竘驒&猣沠鈰j" _迈席b;揠嶄慆h邠?鑘塼u詛扤 筤"7P骀T摧沪鄜)t罡 嵭?,椿〝,+抏(Im辴D壶ov麩=/?D.徫R殉Qn?葴W[杤+ 挂8A?5J愪R)?鑫5註
./m淡誂`狿[8g夸鱽-ぶ徘珘毠犾m浈^饞嚥妲{?[?G/?﹡梲欄9L鎈<禰'?嫒z思衬8容85?Z檙?萑蝚偆覠pr-講?漍趉巿9蛰宰?艍??君"k蹕)谀翙mq?藂.鏦旫鷼庣饵R?9鏣1AWp悗辸mzJ槳鰃棤畫循鐄?楇卋徧?(A攧2Y氃k紖鳉S$餏b〦)j幌7=b塜梢J絡盨庂硺l@ T吀9墕鑈#uU愕 僻!銇阐g炥G\?薎稉娜釰?s乁\鈂w煨羥烔?畯懸€隖?嵯簊輳? E V]Wm]Fy!<&9砎")芝?T〒口-黼鵓R榾-s礒7o碳H穌翛忄/蹵鞊?烀 ?饆闹?擃鷜-凈M?x硬m钦8险%垄A鲗F#;n?c峎鉅n禞4ュ&
棆煇LkGPS潷徧嫮c:H违?眞徵L鵌e>ぇ蠜骳6~7,P!u+ +w滬Wu塻栍d烟岺鋴&齡Q豪偞骻>灼埰钍D ^q懌剄?鐺橬 N_╜u/2?~嵐^?盤W?Jj桼隝篎紛z)虧噇倧宨ztGA蟦k傈?J?=5鍙,藭馶縋[C謤砥"甸MWu锃:?
?郑?Y滤?/瓡鎵屧簖蝽吊?哴:?┩铥|HJ3l缬軌鸗螏ι塴猺kT褼┕I裚+偡KL5澍?韑鍻鑧m瞳?e r暔釼~注Q廦CF昺買€g蔞,!Z?n耑T蝐/軋S謩彲韈前b;`\6鍮趬?謸怷`飧,鯛诶thl喅※v\C髭O載8昕g犅饵戈|E/tcp?c滃番b%elυ雋?[涯厎嵚0Z搰褨=Ad??宬jj9謈!
E糤9*fh旈楉巜3>G(?馔#諠*](娜傟L$D*CE黺眎
T)繧4y閹?礪佇Z駵蒵Bj.%?B`荜L戥阜n%u爔<塭繚Hrは橰IM* 荖6泈厽爦1M諣>lKnu裲少2樄T3?%q+哿>pG*|h'怱笈?v%阐W嫫w箧陷a)搨1u?F5櫁贤u鑎-後柏嘄兽邯p郭晴~庚?瀵鸠x踣呗轎亘s狐tu3籉Z?遄弃莄k皖鎳~蒀啁A终??田6朊辍棸愿?h虀X?蠂鮪{檅RP隥]墚z琽8鼜鮦[n
=23磰鹐:I揑l廂m?郇?)*敂K烤P?2睕 垉舃黛Y颰_辰U蟁{礄7嫲!jBY?4i ?孆 伔5漲(Z垽i遝!5Pm0r%&酽)Ve悎椖L軸雗鍽荂獰;默撞=;?44?]耬?!Q?o聄]詨l盨}蹑湐@,覯?Nv鷸[w?昰`*?留w柲"騒狈z麿呩
$|6枷^b賫(?)沧汃?蟾_d/矾i峼cAkX療?蝣榺檞氺wヅ%@玥??慴蘕r6 ^谣賐┱9?鶡充c` muzs螠m??噥8L嗬]弲.:穅}?屔??`8傖 勻4倐J+7錔-(饦vrTHe)粕6泫憁謎?榟蘷肞?嗶?鋥N?!6a
w涒c(緿h覾1靽B釉↙C钒y?SJ硻G钷薸w氮~?P`!`叩濿!~嗖跴譄?浥5~LV\i€瓬}矑PP{Xb神`螝屝H28鐊$#櫱o"蒆鎼葀[疦/葀eV邷@蚑咜絲鵴? 狝'vP芌?'陮6轫ZF xe濢>O鈴a 磳}
贪5&k8?t?觾鹁@ k?(u .鼯谺拌埯wL]趶7?c?k慍0鋦瑷?連ぴTJ)螾f?pS輧猨?D偤.灿<?諹O⑽OA/0嬲訡凇
緰$廚i劼髠F?i禌#Kヂ狱浥-f€G@b们y膟稧$€-憪衘塂aU锞h塂鰍嘱〥a灧TB钷
(aZzA礝/嶛O3粢獪傼奷?&%欽\慺j鱘钙!渷@?6$U?l饇腝j€A淯c0鱜锌@D坯k0;k啍A楘R剩早~宭檕#&翆錬 cw 〒!皴1庚h韫a c脫廀涳桗0挶?c4;潆綟?剣D憘尶1k{搟L)皡I\厠璅?粏挈喂蕤[%鷓喕膚8棞€嚾奪擾嬇,N珜G?4瞔Y苅?伖話鯩艳詴d凝t瓻t爄鉞q鯷潱茙猱np.?h肜痦篨.p昬磸'饛€{T挪X郯紜
泧鷡=#硛嬥G虨?絒 ょT?扃犓 硑A委'&|ZL'繤cvj眿勍誅cnf 禌鵦GTj灱濣r;?佳晌浐堍J鋦鄒O策蛛m場驦冻乹a,桃% H捣I昮瘖沪?牝
#官V3嚯Y?蜜?p|駸 t'崔
endstream
endobj
45 0 obj
<>stream
H??T萒?54?40W071??Q儒251?55剫 D屚L?0]p憄匋.}鱜C咊b瓸.c=3K 4?510R@讘溗ワ檏む捪 邩
endstream
endobj
187 0 obj
<>stream
H塡NA 繱奡?铑铑?繍;笽&妭 Sf藤
泲qc[莂;;厣.V偻霾忼郹镻1G9苢N捌?墻8蝦庴\(]鼰&椆耈畄茕饭胅顀?L饜GY?Ox?烍倵尖5ox嘶襸>饝O偶/|统饙稂?盔蜔獐 o?
endstream
endobj
181 0 obj
<>stream
H塗忼o 唚~吳?R㏒男玁市5最8)Rc怌嘃]<鰇薱軗O ?尀V淈乯羪泇痁;2妹?渰t潗9?捺pz紀頏颈C?玲?縭`Xc)AZ兠Q茹媺痜F麐澐埿V_韮兠%媗hBコy衻濑鏒{%.}6,畷Ёφ瀵慈躛Q:數nb焓渦州珳"尬C,s j
endstream
endobj
186 0 obj
<>stream
H?脟[蘱礼黜Qw淄nt隫EvVv吿e刣U$2B劉l⑾愤難^ t:璣`岤L&f宅莃裞U瓃l6=v惠@Q憗b地€C:F淣#.Y??QMx?k嘛S黤J赻P?? [(S#Vbj訨燒r0H[k愽韲B=鈽z$鸟ma:诿?妞z,泰N螠坧Vv灁8'蠠杇矢 晃F殛宺Q^:'螱闁=b魌鸥,痋宷U鏩w淾?熙藂n╓芗殸O綎郪o傼?撥緫鋷納S?棺W锡[部9x[蕞`H>?颱餚邨旤?儠<瑶y愨0盆p?鮍奼#i灚徲紉抐\}氭録??r鈟唹奘w泸e嗺r騏柹譟>葟o銬柀熮骧麼}庨I5咸嘃_?j縈鍣?0儡哒/~L?]童氝蛳79[胈鵲 ?<
endstream
endobj
194 0 obj
<>stream
? Adobe d€
s !1AQa"q2憽盉#罵厌3b?r傫%C4S挗瞔s?D'摚?Tdt靡&
剶EFごV覷(蜚竽凿鬳u厱サ耪艴fv問Χ浦骣7GWgw嚄Х亲琪8HXhx垬ǜ蓉桫)9IYiy墮┕少轾*:JZjz姎授犏 m !1AQa"q亼2”裂?BRbr?$4C扴%猜s?釪T
&6E'dtU7颍趁()鱼髣敜茨凿鬳u厱サ耪艴FVfv問Χ浦骣GWgw嚄Х亲琪8HXhx垬ǜ蓉桫9IYiy墮┕少轾*:JZjz姎授犏 ? 鹝鶛瀵瘦S]3^^?]酥K赀j7L>m猱?莈摂?豇鍡,刟d潃胥朓腨=:7鎤缜槦雚窏?琫<搉颉?敿P啩P狦瓜,??榘糖M勪xA鱀~u顉m7铲$/$o雳~]蜰M甼轥蝼鴥? 槖yV閘跤猐(AL裁s??G蒲鯈?^迎礛.郳佥-Q颇?)軾N欒ZmN-N8迮!(HX#?9c憣?y{b瘋 $见{鵮鵌鎚.澶誹翦驩遢湰妖転`綎?濥Tq %fN僬v瀽ju1愗_几 <_棹貤C?蝗稂┹4?批菚?1赎耻L殃z\M眂ポ绽,螌c q犱鮴钧=惻祃寇侎c巀?嬶{呒葭/?丝.k龉c蛁璃巯ft羜u/劲閝咯Mi}=フ觫峴oem箠H銌邍?.宛B傈:\p嶤艱墶bv鍻(麸H朆H騁汓 =鶉^騬iZ?湎-蓦'Z巬Em颾褐-谑典粳rr樂D%璑:|1咾昇@U{円尔Z裎S?“?6繙Z嵽畾艞攖鼽醍铓7梎Y町?Y|锚iSG+┾侄蜊K0e孒腽糒胡"<&g€JR?噦2 LA?[迫??妓gO y蔧2mS顑? 驨沬wo=闯懿