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Rapid detection of duck hepatitis virus type-1 by reverse transcription

Journal of Virological Methods 182 (2012) 76–81

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Journal of Virological

Methods

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j v i r o m e

Rapid detection of duck hepatitis virus type-1by reverse transcription loop-mediated isothermal ampli?cation

Cuiping Song,Hongquan Wan,Shengqing Yu,Xiangan Han,Xusheng Qiu,Qinghai Hu,Lei Tan,Chan Ding ?

Shanghai Veterinary Research Institute,The Chinese Academy of Agricultural Sciences (CAAS),518Ziyue Road,Shanghai 200241,China

Article history:

Received 28December 2011

Received in revised form 8March 2012Accepted 12March 2012

Available online 20 March 2012

Keywords:

Duck hepatitis

Reverse transcription loop-mediated isothermal ampli?cation (RT-LAMP)Sensitivity Speci?city

a b s t r a c t

A reverse transcription loop-mediated isothermal ampli?cation (RT-LAMP)assay for the detection of duck hepatitis virus type-1(DHV-1)was https://www.wendangku.net/doc/e73311437.html,ing primers speci?c to the highly conserved 3D gene of DHV-1,the developed RT-LAMP assay detected the viral RNA of DHV-1extracted from both allantoic ?uid and liver samples of infected ducks.The assay is as sensitive as RT-PCR,and shows no cross-reaction with other common avian viral and bacterial pathogens.In addition to detection via ethidium bromide staining following gel electrophoresis,naked-eye observation after staining with SYBR Green I dye can be used to detect RT-LAMP products;this enables ?eld application of this assay.The ?ndings demonstrate that RT-LAMP can serve as a helpful tool for the detection and surveillance of DHV-1in the poultry industry.

1.Introduction

Duck hepatitis is an acute and highly contagious disease affect-ing ducklings,particularly those under 3weeks of age (OIE,2010).The disease,characterized by hepatitis in infected birds,was ?rst observed in Long Island,New York,in 1945(Levine and Fabricant,1950).With a mortality rate that may be as high as 95%,the disease is of signi?cant economic importance in areas worldwide where ducks are reared.Duck hepatitis virus (DHV),the etiologic agent of duck hepatitis,is a member of the family Picornaviridae .Three types of DHVs,type-1,type-2,and type-3,have been reported,with type-1DHV-1being the most common and widespread internationally (Asplin,1965).Sequence analysis has shown that type-1DHVs are genetically unrelated to other members in the same family.It has therefore been proposed that they be placed in a novel genus,Avi-hepatovirus ,within the family Picornaviridae (Asplin,1965;Kim et al.,2006;Levine and Fabricant,1950;Tseng et al.,2007a ).The genome of DHV-1has a single,long open reading frame (ORF)encoding a polyprotein,which is cleaved into a leader protein,3structural proteins (VP0,VP1,and VP3),and 8non-structural pro-teins (2A1,2A2,2B,2C,3A,3B,3C,and 3D).The 3D protein is an RNA-dependent RNA polymerase (Ding and Zhang,2007;Kim et al.,2006;Rodriguez-Wells et al.,2001;Tseng et al.,2007a,2007b )that

?Corresponding author.Tel.:+862134293441;fax:+862134293461.E-mail address:shoveldeen@https://www.wendangku.net/doc/e73311437.html, (C.Ding).

serves as an important candidate protein in antiviral and differ-ential diagnosis.The 3D gene is highly conserved among DHV-1strains (DNA sequence identity,>97.5%)and has often been used to design primers for molecular diagnosis of DHV-1infection (Jiong-gang et al.,2007).

Several methods have been established for the detection of DHV-1infection,including microneutralization assay,indirect hemagglutination tests,agar gel diffusion precipitation,enzyme-linked immunosorbent assay,and the direct ?uorescence-antibody technique (Fan et al.,1998;Gabridge and Newman,1971;Hwang,1969;Ma et al.,2008;Maiboroda,1972;Murty and Hanson,1961;Sun Quanyun and Li Jinson,1997;Taylor and Hanson,1967;Vertinskii et al.,1968;Zhao et al.,1991).However,these meth-ods are time consuming and lack the desired sensitivity.More recently,RT-PCR and real-time PCR have been established to detect DHV-1(Anchun et al.,2009;Ding Chun-yu,2007;Gao Jun and Jian-hua,2008;Yang et al.,2008).Despite their high sensitivity and speci?city,these methods require expensive equipment and high levels of expertise,factors that have restricted their use in labo-ratories with limited ?nancial resources,particularly those in the ?eld.

Loop-mediated isothermal ampli?cation (LAMP)is a molecular method used to amplify DNA or RNA under isothermal https://www.wendangku.net/doc/e73311437.html,MP is a rapid and highly speci?c assay (Chen et al.,2008;Le Roux et al.,2009;Notomi et al.,2000).In this study,a reverse tran-scription LAMP (RT-LAMP)assay for the detection of DHV-1was established and assessed.The sensitivity and speci?city of the assay were also evaluated.

C.Song et al./Journal of Virological Methods182 (2012) 76–8177

Table1

Viral and bacterial strains in this study.

Strains Year of collection and description Reference or source

FC642009,DHV-1Song et al.(2011)

SH2010,DHV-1wild-type strain

from duck

HQ265433

MY2009,DHV-1wild-type strain

from duck

GU363951

HB2011,DHV-1wild-type strain

from duck

Hu et al.(2011)

AV2111DHV-1ATCC

SY52010,DHV-1wild-type strain

from duck

EF407861

Chicken/Jiangsu/

LWC/2011

AIV JF715043.1

LaSota NDV CVCC

AV1221DPV ATCC

CVCC1547Avian pathogenic Escherichia coli

reference strain

CVCC a

8785Riemerella anatipestifer reference

strain,serotype12

CCUG b

a Chinese Veterinary Culture Collection Center.

b Culture Collection,University of Goteborg,Sweden.

2.Materials and methods

2.1.Viruses and bacteria

The viruses and bacteria used in this study are listed in Table1. Viruses FC64,SH,MY,HB,SY5,AV2111,and AV1221were propa-gated in10-day-old embryonated speci?c pathogen-free(SPF)duck eggs,and viruses chicken/Jiangsu/LWC/2011,La Sota were propa-gated in9-day-old embryonated SPF chicken eggs.Allantoic?uid was stored at?80?C for RNA extraction.Escherichia coli CVCC1547 and Riemerella anatipestifer8785were obtained from the Chinese Veterinary Culture Collection Center(CVCC)or the Culture Collec-tion of the University of Goteborg,Sweden,and they were cultured in Luria–Bertani(LB)and tryptone soy broth(TSB)media under the recommended conditions.

2.2.RNA extraction

Viral RNA was extracted from allantoic?uid or tissue samples with TRIzol reagent(Invitrogen,USA)following the manufacturer’s instructions.Liver samples(0.5g/sample)were homogenized in phosphate-buffered saline(PBS)to a?nal dilution of1:10,and a 200-?l aliquot was processed for RNA extraction.The eluted RNA samples were quanti?ed using an ultraviolet spectrophotometer (1.8

2.3.RT-LAMP reaction

The nucleotide sequences of the DHV-13D gene available from GenBank were analyzed using PrimerExplorer V4soft-ware(http://primerexplorer.jp/elamp4.0.0/index.html)to design the RT-LAMP primers.The outer primers used were F3and the com-plementary sequence of B3.The inner primers included FIP and BIP. The locations,names,and sequences of the primers are shown in Fig.1.

RT-LAMP was carried out in a25-?l reaction volume.The reac-tion mixture consisted of0.2?M each of the F3and B3primers, 0.8?M each of the FIP and BIP primers,0.5M betaine(Sigma, USA),8U Bst DNA polymerase(New England Biolabs,USA),5U avian myeloblastosis virus(AMV RTase;Takara,China),5U RNase inhibitor(Takara,China),2.5?l10×thermal buffer,0.4?M of each dNTP(Promega,USA),4mM MgSO4(Takara,China),0.1%Triton X-100(Sigma,USA),and2?l of viral or tissue RNA.The mixture was incubated at63?C for20,30,40,50,and60min to determine Table2

Clinical liver samples.

Samples Year of collection Reference or source

12001Jiangsu Jiangyin,this study 21995Jiangsu Dongtai,this study 32001Hefei Dashushan,this study 42000Hexian,this study

52002Hefei,this study

62001Jiangsu,this study

72001Wuxi,this study

82001Hefei Yuchang,this study 92001Shandong,this study

102000Zhejiang,this study

AFb12003Shandong,this study

AFb22003Shandong,this study

AFh22003Shandong,this study

142002Anhui,this study

152001Zhejiang,this study

the optimal reaction time.After each incubation time,the reac-tion was stopped by heating at80?C for5min.RT-LAMP products were detected using2%agarose gel electrophoresis with ethidium bromide staining or by visual observation,for which1?l of SYBR Green I dye was added to the reaction tube upon completion of the RT-LAMP reaction.The color change was observed by naked eye observation.The solution was also observed for?uorescence under UV light at365nm.

2.4.Sensitivity and speci?city of RT-LAMP

To evaluate the sensitivity of RT-LAMP,various amounts (0.145?g, 1.45×10?2?g, 1.45×10?3?g, 1.45ng,0.145ng, 14.5pg,1.45pg,and0.145pg)of viral RNA of the DHV-1FC64 strain were used as templates in RT-LAMP assays.To evaluate the cross-reactivity of the3D primer set with other common avian pathogens,six strains of DHV-1(SH,MY,SY5,HB,FC64,and AV2111),avian in?uenza virus(AIV)A/chicken/Jiangsu/LWC/2011 strain(H9N2),Newcastle disease virus(NDV)La Sota strain,duck plaque virus(DPV)AV1221,E.coli CVCC1547,and R.anatipes-tifer8785(Table1)were tested with the RT-LAMP assay.RNAs extracted from all viruses and bacterial cultures were used as RT-LAMP templates.

2.5.Experimental and clinical liver samples

For experimental infection,embryonated SPF duck eggs pur-chased from the Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences(CAAS),were incubated at 37?C.Subsequently,ten1-day-old ducklings were intramuscularly infected with100median lethal doses(LD50)of the DHV-1SH strain (104.58LD50/0.2ml).Another group of10ducklings was injected with PBS(0.2ml/bird)and served as control subjects.The ducklings were monitored for7days to detect the presence of clinical disease. Livers from dead and euthanized infected and control ducklings were collected.Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee(IACUC) guidelines set by the Shanghai Veterinary Research Institute,CAAS.

During the period1995–2003,15liver samples had been collected from sick ducks at duck farms in Anhui,Shandong,Zhe-jiang,and Jiangsu provinces(Table2)and stored at?80?C.These ducks had exhibited clinical signs(ataxia,spastic legs,and an opisthotonus shape)and gross lesions or symptoms(such as liver enlargement and bleeding)characteristic of DHV-1infection.Both experimental and clinical liver samples were tested with the DHV-1 RT-LAMP assay.

78 C.Song et al./Journal of Virological Methods182 (2012) 76–81

ttagctgtattgggctatgacatcaaccaatgttatgtcgttagttatggggatgactgtgttctgtcagtgccagaggtgcgc

3F B2

gatatctcaaaactatcgcactatttcaagcttttctttggtatgacagcaacagctagtgacaaagaaagtgacataacttg g

ctagcccctatggagata gaa tttctgaagagaactcctgcctt

F35’ GC TATGAC ATCAACCA ATG T 3’

B35’ TC TATCTCC ATAGGGGC TAG 3’

’TAGTTTTGAGATATCGCGCACCTCTGTCGTTAGTTATGGGGATGA3’(F1+B1)

FIP:5TAGTTTTGAG ATATCGCGCACCTCTGTCGT TAGT TATGGGG ATGA 3 (F1+B1)

BIP:5’TCGCACTATTTCAAGCTTTTCTTTGGT TATGTCACTTTCTTTGTCAC TAG3’

(F2+B2)

Fig.1.Primers for DHV-1RT-LAMP.The primers were designed based on the3D gene sequences of DHV-1and are indicated by the arrows under the sequence.F3and B3 represent the outer primer pair,while FIP(F1+B1)and BIP(F2+B2)represent the inner primer pair.

2.6.RT-LAMP for DHV-1detection in experimental and clinical tissue samples

All35duck liver samples were analyzed for the presence of DHV-1using RT-LAMP under optimal reaction conditions.To val-idate the RT-LAMP results,RT-PCR(Anchun et al.,2009)was also performed for each of the samples.Brie?y,reverse transcription was performed with a random primer and M-MLV reverse tran-scriptase(Invitrogen,USA).PCR was performed with the primers PF(5 -CCTCAGGAACTAGTCT-3 )and PR(5 -GGAGGTGGTGCTGAAA-3 )to amplify a220-bp fragment of the DHV-13D gene.The PCR was optimized to contain4?l dNTP(2.5mM;Tiangen,China), 2.5?l10×buffer with MgCl2(25mM),1?l of both forward and reverse primers(10?M),1?l of cDNA template,0.5?l of Taq DNA polymerase(5U/?l;Tiangen,China),and distilled water in a?nal volume of25?l.Ampli?cation was carried out under the following conditions:95?C for4min;followed by30cycles of 94?C for30s,52?C for30s,and72?C for30s;and?nal extension at72?C for10min.The ampli?ed DNA fragments were ana-lyzed by electrophoresis in1.5%ethidium bromide-stained agarose gel.

3.Results

3.1.Establishment of an RT-LAMP assay for DHV-1detection

A set of4primers for RT-LAMP were designed on the basis of highly conserved regions of the3D gene of https://www.wendangku.net/doc/e73311437.html,ing the primer set selected,a one-step RT-LAMP assay was developed for the rapid detection of DHV-1.Viral RNA of the DHV-1FC64strain, extracted from infected allantoic?uid,was used for the optimiza-tion of the assay reaction time.The RT-LAMP reaction was carried out at63?C for10,20,30,40,50,and60min.As shown in Fig.2, while there was no signi?cant ampli?cation with reaction times of 10–30min,incubation for40min or longer yielded clear,speci?c products;therefore,40min was used as the optimal reaction time in subsequent assays.

RT-LAMP was also conducted with the addition of SYBR Green I dye.In samples with positive ampli?cation,the original orange color of the reaction mixture changed to green rapidly,whereas in negative control samples without ampli?cation,the mixture retained its orange color.Under UV light,the positive ampli?ca-tion solution displayed a bright?uorescence,whereas the negative solution did not

(Fig.3).Fig.2.Optimization of reaction time of DHV-1RT-LAMP.Different reaction times from10to60min were tested to determine the minimum RT-LAMP reaction time. Lane1,DL2000DNA marker;lanes2–7,reaction times of10,20,30,40,50,and 60min,respectively;

lane8,negative control.

Fig.3.Visual observation of ampli?cation products in DHV-1RT-LAMP.Visual observation,under UV light,of RT-LAMP reaction following addition of SYBR Green I dye.Left,positive sample;right,negative sample.

C.Song et al./Journal of Virological Methods182 (2012) 76–81

Fig.4.Sensitivity of the RT-LAMP assay.A10-fold serial dilution of DHV-1RNA was used to determine the sensitivity of the RT-LAMP https://www.wendangku.net/doc/e73311437.html,ne1,DL5000DNA marker; lanes2–9,ampli?cation products obtained with RNA templates of1.45×10?2?g, 1.45×10?3?g,1.45ng,0.145ng,14.5pg,1.45pg,0.145pg,and0.0145pg,respec-tively;lane10,negative control.

3.2.Sensitivity and speci?city of RT-LAMP

In order to ascertain the DHV-1detection limit of the devel-oped RT-LAMP assay,10-fold serial dilutions of DHV-1strain FC64 viral RNA were tested.As shown in Fig.4,RT-LAMP with1.45pg of viral RNA resulted in clear,speci?c ampli?cation.RT-LAMP using a0.145pg viral RNA template yielded detectable products,but the results were not as clear as those from assays using

more Fig.5.Speci?city of the RT-LAMP assay.RT-LAMP products ampli?ed from RNA extracted from DHV-1strains(lanes2–7:strains AV2111,MY,FC64,SH,SY5,and HB,respectively)showed characteristic ladder-like patterns,while those from tem-plates of other pathogens(lanes8–12:DPV,strains NDV,AIV,R.anatipestifer,and E.coli)did https://www.wendangku.net/doc/e73311437.html,ne1,DL2000DNA marker;lane13,negative control.

template.When0.0145pg of viral RNA was used,the RT-LAMP did not produce any detectable products.Thus,the detection limit of the DHV-1RT-LAMP assay is1.45pg viral RNA.

In order to evaluate the speci?city of the DHV-1RT-LAMP assay, avian pathogens AIV,NDV,DPV,E.coli,and R.anatipestifer were pro-cessed using RT-LAMP,and the results were compared with those obtained with ampli?cation of various DHV-1strains.While the 6DHV-1strains tested all exhibited positive ampli?cation in the assay,none of the other pathogens tested yielded positive results, nor the negative control(Fig.5).These results indicate the high speci?city of RT-LAMP for the detection of DHV-1.

3.3.RT-LAMP of experimental and clinical samples

To con?rm the applicability of the RT-LAMP assay for the detec-tion of DHV-1in?eld-obtained samples,the assay was used for the detection of DHV-1in liver samples from experimentally

infected Fig.6.Experimental samples testing by RT-LAMP and RT-PCR.Twenty experimental liver samples were tested by RT-LAMP and RT-PCR.Panels A and C,RT-LAMP products from the experimental https://www.wendangku.net/doc/e73311437.html,ne M,DL2000DNA marker;lanes1–10,RT-LAMP products from the10experimental and control samples.Panels B and D,RT-PCR products. RT-PCR was carried out to validate viral https://www.wendangku.net/doc/e73311437.html,ne1,DL2000DNA marker;lanes2–11,RT-PCR products from the10experimental and control samples.

80 C.Song et al./Journal of Virological Methods 182 (2012) 76–

Fig.7.Clinical samples tested by RT-LAMP and RT-PCR.Fifteen clinical liver samples were assessed for DHV-1detection using RT-LAMP and RT-PCR.Panel A,RT-LAMP products;lane 1,DL2000DNA marker;lanes 2–16,RT-LAMP products from the 15clinical samples.Panel B,RT-PCR products.From the 15clinical samples,the virus was isolated using embryonated duck eggs;RT-PCR was carried out to validate the presence of the virus in the allantoic ?https://www.wendangku.net/doc/e73311437.html,ne 1,DL2000DNA marker;lanes 2–16,RT-PCR products from the 15clinical samples.

ducks.All the 10infected liver samples yielded positive results by the RT-LAMP assay,while all the negative control samples produced negative results (Fig.6,panels A and C).To validate these results,RT-PCR was performed on the same samples (Fig.6,panels B and D).The RT-PCR data were consistent with the RT-LAMP results.Of the 15clinically obtained samples,14yielded positive DHV-1RT-LAMP results (Fig.7,Panel A).Subsequent RT-PCR analysis of the same 14samples produced positive results for the presence of DHV-1(Fig.7,Panel B).These results suggest that the DHV-1RT-LAMP assay is as sensitive as RT-PCR for the detection of DHV-1.

4.Discussion

LAMP is a novel method of nucleic acid ampli?cation and detec-tion ?rst reported by Notomi et al.(2000).The LAMP assay,which employs DNA polymerase and a set of primers that recognize up to 6distinct sequences on the target DNA,is rapid and simple to perform.In recent years,the LAMP assay has been modi?ed and applied in the diagnosis of many viral diseases (Chen et al.,2008;Fukuta et al.,2003;Lakshmi et al.,2008;Le Roux et al.,2009;Liu et al.,2011;Shi et al.,2010;Wang et al.,2011;Yang et al.,2011).In the present study,a one-step RT-LAMP assay was established for the detection of DHV-1,an important pathogen in the poultry industry.

The primers used in this study were designed speci?c to the 3D gene,which is highly conserved among DHV-1strains (Ding and Zhang,2007).The two sets of primers (outer and inner)ampli-?ed the 3D structural glycoprotein sequence of the 3D gene.The optimal conditions for the RT-LAMP reaction for the detection of DHV-RNA were found to be 63?C and 40min.In this study,the DHV-1detection limit of RT-LAMP was found to be 1.45pg of viral RNA.In the detection of DHV-1from both experimental and clin-ical samples,the RT-PCR results were consistent with those from RT-LAMP,which suggests that the RT-LAMP assay is as sensitive as an assay using RT-PCR.Thus,the suitability of using the RT-LAMP assay for detection of DHV-1RNA in infected samples was successfully con?rmed.The speci?city of the LAMP reaction was extremely high because it used 4primers that recognize 6distinct

regions in the target RNA.In this study,all other avian pathogens,including AIV,NDV,DPV,E.coli ,and R.anatipestifer ,yielded neg-ative RT-LAMP results,indicating the high speci?city of this assay for DHV-1detection.

The entire duration of the RT-LAMP assay,including RNA extrac-tion,the reaction,and the ?nal visualization of ampli?cation products,was less than 2h,while at least 5–6h is needed for a conventional RT-PCR-based method.This suggests that RT-LAMP is a more rapid method for the detection of DHV-1than the RT-PCR method.In addition,RT-LAMP can be performed with commonly available and inexpensive equipment.The RT-LAMP reaction is exe-cuted in a single tube and only requires a simple water bath to provide a constant temperature of 63?C for 40min;moreover,the ampli?cation products can be detected by naked eye observation.Because of these advantages,the RT-LAMP assay is considerably more applicable than RT-PCR,particularly in the ?eld.

With its high sensitivity,speci?city,rapidity,and ease of visual-ization of the ?nal products,the RT-LAMP assay established in this study will serve as a useful method for the detection and surveil-lance of DHV-1.

Acknowledgments

This work was funded by Central Public Welfare Research Insti-tutions and Basic Scienti?c Research Business Expenses (2010JB02),Chinese Special Fund for Agro-scienti?c Research in the Public Interest (201003012)and Chinese National High-tech R&D Program (863Program,2011AA10A209).

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经典的英语歌曲歌词大全

歌曲：pretty boy I lie awake at night 晚上我躺在床上没有一点睡意 See things in black and white 世界对我来说只有黑与白 I've only got you inside my mind 你的形象无时无刻不在我脑中闪动 You know you have made me blind 我的眼里只有你 I lie awake and pray 我清醒地躺在床上祈祷 that you would look my way 祈祷你会看见我 I have all this songing in my heart 我的心中充满渴望 I knew it right from the start 一开始我就知道 Oh my pretty pretty boy I love you 我的漂亮男孩我爱你 Like I never ever loved no one before you 在你之前我从未这样爱过一个人

Pretty pretty boy of mine 我的漂亮男孩 Just tell me you love me too 告诉我你也爱我 Oh my pretty pretty boy I need you 我的漂亮男孩我需要你 Oh my pretty pretty boy I do 我的漂亮男孩，是真的 Let me inside make me stay right beside you 让我进来让我留在你身旁 I used to write your name 我曾写下你的名字 And put it in a frame 并把它框起来 And sometimes I think I hear you call Right from my bedroom wall 有时我觉得我在我房间里听到了你的呼唤 You stay a little while 你待了一会儿

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1 Twinkle Twinkle Little Star Twinkle, twinkle, little star, How I wonder what you are. Up above the world so high, Like a diamond in the sky. Twinkle, twinkle, little star, How I wonder what you are! When the blazing sun is gone, When there's nothing he shines upon, Then you show your little light, Twinkle, twinkle, all the night. twinkle, little star, How I wonder what you are! Twinkle, twinkle, little star, How I wonder what you are! 2 Finger Family Daddy finger, Daddy finger, where are you? Here I am. Here I am. How do you do? Mammy finger, Mammy finger, where are you? Here I am. Here I am. How do you do? Brother finger, Brother finger, where are you? Here I am. Here I am. How do you do? Sister finger, Sister finger, where are you? Here I am. Here I am. How do you do? Baby finger, Baby finger, where are you? Here I am. Here I am. How do you do? 3 Ten Little Indian Boys One little, two little, three little Indians, four little, five little, six little Indians, seven little, eight little, nine little Indians, ten little Indian boys. Ten little, nine little, eight little Indians, seven little, six little, five little Indians,

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雪绒花（Edelweiss）》、《Do-Re-Mi》、《you are my sunshine》这三首英文歌词很多人都认为是最好听的儿童英文歌曲，无论是歌词还是旋律都健康有意义，也很好听。下面是这三首儿童英文歌曲的歌词《雪绒花（Edelweiss）》 edelweiss, edelweiss every morning you greet me small and white, clean and bright you look happy to meet me blossom of snow may you bloom and grow bloom and grow forever edelweiss, edelweiss bless my homeland forever small and white, clean and bright you look happy to meet me blossom of snow may you bloom and grow bloom and grow forever edelweiss, edelweiss bless my homeland forever

《Do-Re-Mi》 doe a deer a female deer ray a drop of golden sun me a name i call myself far a long long way to run sew a needle pulling thread la a note to follow sew tea a drink with jam and bread that will bring us back to do doe a deer a female deer ray a drop of golden sun me a name i call myself far a long long way to run sew a needle pulling thread la a note to follow sew tea a drink with jam and bread that will bring us back to do do-re-mi-fa-so-la-te do-so-do 《you are my sunshine》 You make me happy when skies are grey You'll never know, dear, how much I love you

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. Twinkle Twinkle Little Star 1 Twinkle, twinkle, little star, How I wonder what you are. Up above the world so high, Like a diamond in the sky. Twinkle, twinkle, little star, How I wonder what you are! When the blazing sun is gone, When there's nothing he shines upon, Then you show your little light, Twinkle, twinkle, all the night. twinkle, little star, How I wonder what you are! Twinkle, twinkle, little star, How I wonder what you are! Finger Family 2 Daddy finger, Daddy finger, where are you? Here I am. Here I am. How do you do? Mammy finger, Mammy finger, where are you? Here I am. Here I am. How do you do? Brother finger, Brother finger, where are you? Here I am. Here I am. How do you do? Sister finger, Sister finger, where are you? Here I am. Here I am. How do you do? Baby finger, Baby finger, where are you? Here I am. Here I am. How do you do? 3 Ten Little Indian Boys One little, two little, three little Indians, four little, five little, six little Indians, seven little, eight little, nine little Indians, ten little Indian boys. Ten little, nine little, eight little Indians, seven little, six little, five little Indians, four little, three little, two little Indians, . .

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WaitDI 等待一个输入信号状态为设定值 WaitDO 等待一个输出信号状态为设定值 3. 程序注释指令说明 Comment 对程序进行注释 4. 程序模块加载指令说明 Load 从机器人硬盘加载一个程序模块到运行内存 UnLoad 从运行内存中卸载一个程序模块 Start Load 在程序执行的过程中，加载一个程序模块到运行内存中 Wait Load 当Start Load使用后，使用此指令将程序模块连接到任务中使用CancelLoad 取消加载程序模块 CheckProgRef 检查程序引用 Save 保存程序模块 EraseModule 从运行内存删除程序模块 5. 变量功能指令说明 TryInt 判断数据是否是有效的整数功能说明 OpMode 读取当前机器人的操作模式 RunMode 读取当前机器人程序的运行模式 NonMotionMode 读取程序任务当前是否无运动的执行模式 Dim 获取一个数组的维数 Present 读取带参数例行程序的可选参数值 IsPers 判断一个参数是不是可变量 IsVar 判断一个参数是不是变量 6. 转换功能指令说明 StrToByte 将字符串转换为指定格式的字节数据 ByteToStr 将字节数据转换为字符串 5.6.3 运动设定 1. 速度设定功能说明 MaxRobSpeed 获取当前型号机器人可实现的最大TCP速度

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128首英文儿歌歌词

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0 5 10 zer o fiv e ten, zer o fiv e ten, ten fiv e zer o, ten fiv e zer o, zer o fiv e ten, zer o fiv e ten, clap your hands and come again, one two three four five six seven eight nine ten, one two three four five six seven eight nine ten, zer o fiv e ten, zer o fiv e ten, ten fiv e zer o, ten fiv e zer o, zer o fiv e ten, zer o fiv e ten, clap your hands and come again, one two three four five six seven eight nine ten, one two three four five six seven eight nine ten, abc song a b c d e f g, h I j k, l m n o p, q r s, t u v, w x, y and z, now I know my a b c, tell me what you think of me drive a car I can,I can drive a car, Drive, drive, drive a car, I can ride a bicycle, Ride, ride, ride ride ride, I can drive and you can ride, you can ride and I can drive. I can,I can drive a car, Drive, drive, drive a car, I can ride a bicycle, Ride, ride, ride ride ride, I can drive and you can ride, you can ride and I can drive. finger family daddy finger，daddy finger，

经典英文儿歌及歌词

歌曲和儿歌附录去打猎我们去打猎，我们去打猎。嘿哟，马里奥。我们要去打猎。 A Hunting We Will Go A hunting we will go, a hunting we will go, Hi-ho the merrio, a hunting we will go. 拍拍你的小手拍拍你的小手，手心在一起。拍拍你的小手，把手放一起。歌词还可以改为：(2)摸摸你的鼻子(3)拍拍你的膝盖(4)拍拍你的脑袋 Clap, Clap, Clap Your Hands Clap, clap, clap your hands, Clap your hands together. Clap, clap, clap your hands, Clap your hands right now. Additional verses: (2)Touch your nose(3)Tap your knees(4)Pat your head 你看到过一个女孩吗你看到过一个女孩吗，一个女孩，一个女孩？你看到过一个女孩从这里走过吗？从这里走过，从这里走过。你看到过一个女孩从这里走过吗？

Did You Ever See a Lassie (or Laddie) Did you ever see a lassie, a lassie, a lassie? Did you ever see a lassie go this way and that? Go this way and that way, go this way and that way. Did you ever see a lassie go this way and that? 小蜘蛛小蜘蛛顺着水管爬上去，可是雨水把它冲了下来！太阳出来了，水没有了，小蜘蛛又可以顺着水管爬上去！ The Eensy Weensy Spider The eensy weensy spider went up the water spout, Down came the rain and washed the spider out. Out came the sun and dried up all the rain And the eendy weensy spider went up the spout again. 头，肩膀，膝盖和脚趾头，肩膀，膝盖和脚趾，膝盖和脚趾。头，肩膀，膝盖和脚趾，膝盖和脚趾。眼睛，耳朵，嘴巴和鼻子。头，肩膀，膝盖和脚趾，膝盖和脚趾。 Head and Shoulders, Knees and Toes Head and shoulders, knees and toes, knees and toes. Head and shoulders, knees and toes, knees and toes. Eyes and ears and mouth and nose.

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