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1.寵物病原體

1.1貓冠狀病毒熒光RT-RAA檢測方法的建立

1.2基于等溫?cái)U(kuò)增技術(shù)的布魯氏桿菌檢測方法及試劑盒研究

2.禽類疫病

2.1Establishment of reverse transcription re combi-nase-aided amplification-lateral-flow dipstick andreal-time fluores cence-based reverse transcriptionrecombinase-aided amplification methods for detec-tion of the Newcastle disease virus in chickens

2.2RAA-LFD assay-a specific and sensitive method forvisual detection of avian infec tious laryngotracheitisvirus

2.3Reverse transcription recombinase-aided amplifica-tion assay combined with a lateral flow dipstick fordetection of avian infectious bronchitis virus

2.4Research Note: Rapid detection of avian infectiouslaryn gotracheitis virus with real-time fluores-cence-based recombinase-aided amplification

2.5Reverse-transcription recombinase-aided amplification assay for H7 subtype avian influenza virus

2.6重組酶介導(dǎo)的等溫?cái)U(kuò)增法檢測H7N9禽流感病毒

2.7禽流感RT-RAA側(cè)向流試紙條可視化檢測方法的建立

2.8多殺性巴氏桿菌熒光RAA快速檢測方法的建立

2.9禽流感病毒H5N1的重組酶介導(dǎo)檢測方法學(xué)研究

3.動(dòng)物疫病

3.1Clinical Validation of Two Recombinase-Based lsothermal Amplification Assays (RPA/RAA)for the RapidDetection of African Swine Fever Virus

3.2非洲豬瘟病毒實(shí)時(shí)熒光RAA檢測方法的建立

3.3豬附紅細(xì)胞體重組酶介導(dǎo)等溫?cái)U(kuò)增技術(shù)RAA熒光檢測方法的建立

3.4牛病毒性腹瀉病毒1型RT-RAA快速診斷方法的建立

3.5豬圓環(huán)病毒4型實(shí)時(shí)熒光RAA檢測方法的建立

3.6重組酶介導(dǎo)的油菜莖基潰瘍病菌熒光核酸擴(kuò)增檢測體系的建立

4.植物疫病

4.1CRISPR-Cas Detection Coupled with lsothermalAmplification of Bursaphelenchus xylophilus

貓冠狀病毒熒光ＲＴ－ＲＡＡ檢測方法的建立

摘要根據(jù)貓冠狀病毒(FCoV)7b基因序列，設(shè)計(jì)引物及探針，建立了FCoV熒光RT一RAA檢測方法。該方法能夠在39℃恒溫條件下20min內(nèi)對(duì)病原進(jìn)行快速檢測，與貓皰疹病毒、貓杯狀病毒、貓細(xì)小病毒均無交叉反應(yīng)；對(duì)病毒核酸的最低檢測限為1.97×101fg/uL。用套式PCR與建立的熒光RT一RAA對(duì)30份疑似FCoV感染臨床樣品進(jìn)行檢測，兩種方法符合率為93.33%。結(jié)果表明，建立的FCoV熒光RT一RAA方法適用于FCoV快速檢測。

關(guān)鍵詞：貓冠狀病毒；熒光RT—RAA；快速檢測。

基于等溫?cái)U(kuò)增技術(shù)的布魯氏桿菌檢測方法及試劑盒研究

摘 要：介紹了布魯氏菌病的危害及檢測技術(shù)，研究了布魯氏桿菌等溫擴(kuò)增技術(shù)，說明了其可用于檢測布魯氏桿菌 DNA含量較低的樣品，研制的試劑盒可在30℃～42℃下進(jìn)行等溫?cái)U(kuò)增檢測，5～20min完成 檢測。指出了試劑盒具有快速、靈敏、準(zhǔn)確、重復(fù)性好的優(yōu)點(diǎn)，適合基層和現(xiàn)場檢測，具有良好的應(yīng)用前景。

關(guān)鍵詞：等溫?cái)U(kuò)增；布魯氏桿菌；檢測方法；試劑盒。

Establishment of reverse transcription re-combinase-aided amplification-lateral-flowdipstick and real-time fluorescence-basedreverse transcription recombinase-aidedamplification methods for detection oftheNewcastle disease virus in chickens

ABSTRACT Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection,which does great harm to the poultry industry all over the world.To diagnose the diseasesimply and quickly,2 detection methods were established based on reverse transcription recombinaseaidedamplification(RT-RAA) technology.One is reverse transcription recombinase-aided amplification lateral flowdipstick(RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick;the other is real-time fluores-cence-based reverse transcription recombinase-aided amplification(RF-RT-RAA) that is the combination ofRT-RAA and exo probe.In this study,the reaction conditions such as reaction temper ature and reaction timeof the 2 methods were opti mized,and their specificity and sensitivity were tested.The results showed that theRT-RAA-LFD method could be used to complete reaction within 23 min,and its lowest detectable limit was 102copies/mL,10 times higher than that of the conventional PCR method (103copies/mL);the RF-RT-RAA methodcould be used to complete reaction within 26 min, and its lowest detect able limit was 10 copies/mL,100 timeshigher than that of conventional PCR method (103 copies/mL),and it was as sensitive as real-time fluores-cence-based quantitative PCR (10 copies/mL).The 2 methods had no cross reac tion to the nucleic acid ofother avian pathogens and showed good specificity.A total of 86 clinical samples suspected of the Newcastledisease virus were tested by conventional PCR, real-time fluorescence-based quan titative PCR,RT-RAA-LFD,and RF-RT-RAA.Based on the commonly used conventional PCR method,the other 3 detection methods had acoincidence rate of higher than 93%.In summary,RT-RAA-LFD and RF RT-RAA had high specificity,sensitivity.and efficiency,which were suitable for clinical and laboratory diagnosis,respectively,and provided technicalsupport for the prevention and control of Newcastle disease.

Key words:Newcastle disease virus,reverse transcription recombinase-aided amplification-lateral flowdipstick,real-time fluorescence-based-reverse transcription recombinase-aided amplification,detection.

RAA-LFD assay-a specific and sensitivemethod for visual detection of avian infec-tious laryngotracheitis virus

ABSTRACT The purpose of this study was to explore a specific,simple and sensitive method for diagnosis ofavian infectious laryngotracheitis virus (LTV).The recombinase-aided amplification (RAA) and lateral flowdipstick(LFD) were combined for labelling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the5' end of downstream primer with biotin,respectively.By optimizing the reaction time,temperature andprimer concentration of RAA,a RAA-LFD assay which could be used for detection of infectious laryngotra-cheitis (ILT) was established.After specificity and sensitivity test,the target gene fragments could be ampli-fied by RAA-LFD assay in 20 min under isothermal conditions (37 口),and the amplification products could bevisually observed and determined by LFD within 3 min.There was no cross reaction with nucleic aids of otheravian pathogens,the lowest detectable limit (LDL) of RAA-LFD was 102 copies/uL,and the sensitivity of thismethod was 100 times higher than that of conventional CR with LDL of 104 copies/uL.The results showedthat RAA-LFD assay was highly sensitive,easy to use,and more suitable for clinical detection.

Key words:avian infectious laryngotracheitis virus;recombinase-aided amplification;lateral flow dipstick.