TWI532844B - Method of distinguishing dog coccidia - Google Patents
Method of distinguishing dog coccidia Download PDFInfo
- Publication number
- TWI532844B TWI532844B TW102100134A TW102100134A TWI532844B TW I532844 B TWI532844 B TW I532844B TW 102100134 A TW102100134 A TW 102100134A TW 102100134 A TW102100134 A TW 102100134A TW I532844 B TWI532844 B TW I532844B
- Authority
- TW
- Taiwan
- Prior art keywords
- sample
- nucleic acid
- pcr
- results
- babesia
- Prior art date
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本發明係關於一種區分動物病原微生物的方法,特別是關於一種區分犬焦蟲症分型的方法。 The present invention relates to a method of distinguishing animal pathogenic microorganisms, and more particularly to a method for distinguishing the classification of canine echinococcosis.
隨著少子化、高齡化的趨勢,愈來愈多的寵物進入家庭,成了現代人最重要的「伴侶動物」。據統計,粗估一年台灣寵物相關市場高達250億元,寵物量增多相對於獸醫的需求也越來越多,動物醫學顯得日益重要。 With the trend of declining birth and aging, more and more pets have entered the family and become the most important "companion animals" of modern people. According to statistics, it is estimated that the pet-related market in Taiwan is as high as 25 billion yuan a year. The increase in pets is more and more demanding than veterinarians. Animal medicine is becoming increasingly important.
目前寵物狗的比例占了整體寵物的一半以上,隨著養寵物人口的增加,獸醫院的患病率也隨之提升。由於寵物在被某些病原菌感染後,易有危害身體器官組織,甚至嚴重導致死亡的情況產生,因此把握疾病診斷的黃金期極為重要,獸醫市場對於快速檢測的需求也極為迫切。針對病原菌的檢測在人醫系統上已建立良好的快篩或快速檢驗模式,但在獸醫系統的建立並不完善,現階段僅針對少數貓狗的病原菌開發出快篩,如狗的四合一快篩套組(心絲蟲、萊姆病病原、犬型/馬型艾利希體、血小板型艾利希體)、貓的三合快篩(貓免疫不全病毒、貓白血病病毒、心絲蟲),因此陸續建立其他犬貓傳染性病原菌的快篩試劑套組或快速檢驗模式已是現今獸醫系統的重點。 At present, the proportion of pet dogs accounts for more than half of the total pets. As the population of pets increases, the prevalence of veterinary hospitals also increases. Since pets are susceptible to damage to body tissues and even deaths after being infected by certain pathogens, it is extremely important to grasp the golden period of disease diagnosis. The veterinary market is also in urgent need for rapid detection. For the detection of pathogenic bacteria, a good fast screening or rapid inspection mode has been established in the human medical system, but the establishment of the veterinary system is not perfect. At this stage, only the pathogens of a few cats and dogs are developed, such as the dog's four-in-one. Fast screening kit (heartworm, Lyme disease, canine/horse type Ehrlich, platelet type Ehrlich), cat's triple play (cat immunodeficiency virus, feline leukemia virus, silk) Therefore, the rapid screening kit or rapid test mode for establishing other infectious diseases of dogs and cats has become the focus of today's veterinary system.
焦蟲症是一種血液方面的疾病,由血液寄生蟲所引起,這種寄生蟲稱為焦蟲,是一種「原蟲」,它會導致狗的進行性的貧血。傳播媒介主要是壁蝨(Tick)叮咬,也有少部分是因為輸血及經由胎盤感染給胎兒。 Coccidiosis is a blood-related disease caused by blood parasites, called parasites, a "protozoa" that causes progressive anemia in dogs. The media is mainly Tick bites, and a small part is due to blood transfusions and infections through the placenta to the fetus.
感染後出現症狀大概需要10-20天左右,初期常沒有顯著的症狀,僅會缺乏食慾,然紅血球細胞的主要功能是攜帶氧氣,如果被寄生蟲感染則攜氧功能會降低,缺氧的結果導致精神狀況逐漸變差,嚴重造成自體白血球的吞噬細胞將自身的紅血球吞噬。犬隻在感染後出現的反應與症狀不同,主要分為非特異性、慢性與急性感染。非特異性感染的時候症狀較多,會 有腹水、腸胃道症狀、神經症狀、周邊水腫以及心肺疾病。慢性感染時狗會有間斷性的發燒、食慾不振與體重減輕;而急性感染會有體溫上升、黏膜蒼白、血斑、黃疸、肝脾腫大、甚至急性死亡。 Symptoms after infection may take about 10-20 days. There are often no obvious symptoms in the initial stage. There is only a lack of appetite. However, the main function of red blood cells is to carry oxygen. If infected by parasites, the oxygen-carrying function will decrease. This leads to a gradual deterioration of mental status, which causes phagocytic cells of autologous white blood cells to devour their own red blood cells. Dogs have different responses and symptoms after infection, and are mainly classified into non-specific, chronic and acute infections. Non-specific infection, more symptoms, will There are ascites, gastrointestinal symptoms, neurological symptoms, peripheral edema, and heart and lung disease. In chronic infection, dogs have intermittent fever, loss of appetite and weight loss; acute infections may have elevated body temperature, pale mucous membranes, blood spots, jaundice, hepatosplenomegaly, and even acute death.
目前已有超過一百多種的焦蟲,感染狗的主要分為大、小焦蟲兩型,大焦蟲又名犬焦蟲(Babesia canis),小焦蟲又名吉布松焦蟲(Babesia gibsoni)。當發生焦蟲感染的時候,通常只會出現其中一種,不是大焦蟲就是小焦蟲,很少有混合感染的情形,由於在用藥上有差異,因此需要小心且快速的鑑別,以免延誤病情,造成後續難以根治,且有復發可能。焦蟲的檢測目前尚無快篩或快速檢測方法,因此開發快速與精確的檢測技術是勢在必行。 At present, there are more than one hundred kinds of cokeworms. The infected dogs are mainly divided into two types: large and small worms. The large worms are also known as Babesia canis . The small worms are also known as Gibbs pine worms. Babesia gibsoni ). When a coccidiosis infection occurs, usually only one of them occurs. It is not a large cokeworm or a small cokeworm. There are few mixed infections. Because of differences in medication, careful and rapid identification is required to avoid delaying the disease. , causing subsequent hard to cure, and there may be recurrence. There is no fast screening or rapid detection method for the detection of coccidia, so it is imperative to develop rapid and accurate detection technology.
傳統的焦蟲檢測方式是以血液抹片檢查,不論是大焦蟲或是小焦蟲都會在紅血球內的黑點周邊出現特異性半透明指環,通常在紅血球中發現一顆黑點代表Babesia gibsoni感染,而雙梨形黑點則是感染Babesia canis的特徵。然以此法發現病原的機率並不高,一般都是發病一段時間,焦蟲數目累積一定數量時才看得見。 The traditional method of detecting insects is to use blood smears. Whether it is large worms or small worms, specific translucent rings appear around the black spots in the red blood cells. Usually, a black dot is found in the red blood cells to represent Babesia gibsoni. Infection, and the double pear-shaped black spots are characteristic of Babesia canis infection. However, the probability of finding pathogens in this way is not high, generally it is a period of onset, and it is only visible when the number of coccidia accumulates a certain amount.
早期的焦蟲感染較難從血液抹片中發現,而以傳統的PCR技術檢測,其引子(primer)可能與非焦蟲的DNA(亦即病患自己的DNA)結合,造成偽陽性的問題產生,該領域之習知技藝者已有人將Nested PCR(巢式PCR)技術應用於焦蟲檢測上。Nested PCR主要是利用兩對primer進行兩輪PCR擴增反應。第一輪的擴增與傳統PCR無異,第一輪PCR後的產物再以第二對primer進行專一性的擴增,藉由兩次PCR擴增,降低了複製到焦蟲以外DNA的機率,偵測靈敏度提升了102~103倍,但因為2次PCR易有交叉感染,仍無法做到精準分型與避免偽陽性的產生。 Early coccidiosis infections are more difficult to find from blood smears, and traditional PCR techniques detect that their primers may bind to non-coke DNA (ie, the patient's own DNA), causing false positives. The production of Nested PCR (nested PCR) technology has been applied to the detection of coccidia by those skilled in the art. Nested PCR mainly uses two pairs of primers for two rounds of PCR amplification reactions. The first round of amplification is no different from traditional PCR. The product after the first round of PCR is specifically amplified by the second pair of primers, and the probability of replication to DNA other than the coccidia is reduced by two PCR amplifications. The detection sensitivity has increased by 10 2 ~ 10 3 times, but because of the cross-infection of 2 PCRs, it is still impossible to accurately classify and avoid false positives.
Nested PCR的缺點是實驗操作步驟繁瑣,PCR後無法直接看到結果,需以電泳進一步確認有無焦蟲的cDNA存在,且因為大、小焦蟲的18S rDNA基因序列太過相近,僅有某幾個鹼基不同,因此雖然設計2組不同的primer進行區分,在電泳圖上不一定可以從條帶看出分型,還需以核酸定序儀或 是限制酶RFLP確認,因此至少需要一天以上才能得到檢驗報告。在耗時與精確度尚可的情況之下,有必要發展更快速與更精確的檢測方式來提升焦蟲檢驗的品質。 The disadvantage of Nested PCR is that the experimental procedure is cumbersome. The result cannot be directly seen after PCR. It is necessary to further confirm the presence or absence of the cDNA of the cokeworm by electrophoresis, and because the 18S rDNA gene sequences of the large and small worms are too close, only a few The bases are different, so although two different sets of primers are designed to distinguish, it is not necessary to see the type on the electropherogram, and a nucleic acid sequencer or It is a restriction enzyme RFLP confirmation, so it takes at least one day to get an inspection report. In the case of time-consuming and accurate, it is necessary to develop faster and more accurate detection methods to improve the quality of the coroner test.
承前所述,考量到市場的需求以及現今檢測技術的問題,亟需建立一種快速且精確的犬焦蟲症分型檢測技術,以滿足獸醫診斷上時效性的需求,此技術概念的建立也方便日後在獸醫系統上其他病原菌快速檢測模式的仿效。 As mentioned above, considering the needs of the market and the problems of today's detection technology, it is urgent to establish a fast and accurate dog pyrotype detection technology to meet the needs of veterinary diagnosis timeliness. Other models of rapid detection of pathogens on the veterinary system will follow suit.
緣此,本發明提供一種區分犬焦蟲症分型的方法,其步驟包括:(a)提供一核酸樣本;(b)對該核酸樣本進行一定量即時聚合酶鏈鎖反應(Q real-time PCR);以及(c)進行一熱熔解曲線(melting curve)分析以區分該核酸樣本之犬焦蟲症分型;其中該犬焦蟲症分型包括犬焦蟲(Babesia canis,BC)及吉布松焦蟲(Babesia gibsoni,Bg),且該即時定量聚合酶連鎖反應所使用之引子對SEQ ID NO:3及SEQ ID NO:4(BCBghsp231F/R)係針對該核酸樣本中編碼熱休克蛋白70基因之區段。 Accordingly, the present invention provides a method for distinguishing a dog phobia typing, the steps comprising: (a) providing a nucleic acid sample; (b) performing a certain amount of immediate polymerase chain reaction on the nucleic acid sample (Q real-time) PCR); and (c) performing a melting curve analysis to distinguish the canine worm typing of the nucleic acid sample; wherein the canine genus classification includes Babesia canis (BC) and Kyrgyzstan Babesia gibsoni (Bg), and the primer used in the real-time quantitative polymerase chain reaction pair SEQ ID NO: 3 and SEQ ID NO: 4 (BCBghsp231F/R) for encoding heat shock proteins in the nucleic acid sample A segment of the 70 gene.
較佳地,其中步驟(c)更包括進行一高解析熱熔解(High-Resolution Melting,HRM)分析。 Preferably, wherein step (c) further comprises performing a high-resolution melt-melting (HRM) analysis.
較佳地,其中該核酸樣本係萃取自一個體之血液樣本;較佳地,該血液樣本係為經過血球血漿分離後之血球樣本。 Preferably, the nucleic acid sample is extracted from a blood sample of a body; preferably, the blood sample is a blood cell sample after separation by blood cell plasma.
較佳地,其中步驟(C)更包括使用LC格林(LC green)染劑、賽巴格林(SYBR green)染劑或賽多9(SYTO 9)染劑。 Preferably, wherein step (C) further comprises using LC green dye, SYBR green dye or SYTO 9 dye.
經由本發明所採用的技術手段,以犬焦蟲與吉布松焦蟲之熱休克蛋白(heat shock protein)70基因進行高解析熱熔解分析(high resolution melting)方式可成功地將兩種蟲體進行區分,並利用已知濃度陽性對照組之Ct值求得待測蟲體濃度(可達絕對定量),提供一種快速的檢測方式(包含核酸萃取及 HRM上機時間,共約四小時),大大提供了臨床獸醫師所需之治療黃金時間。在未來可利用本發明所揭露之技術內容及其優點,包括提升檢測精確度、降低偽陽性發生之優點,據以建立大小焦蟲的快速檢測系統,提供更有效率的檢測服務。 Through the technical means adopted by the present invention, two kinds of worms can be successfully performed by high resolution melting method using the heat shock protein 70 gene of canine worm and Gibbs pine worm. Distinguish and use the Ct value of the positive control group with known concentration to determine the concentration of the test insect (up to absolute quantitation), providing a rapid detection method (including nucleic acid extraction and The HRM time (a total of about four hours) greatly provides the prime time for the treatment of clinical veterinarians. In the future, the technical contents and advantages thereof disclosed in the present invention can be utilized, including improving the detection accuracy and reducing the advantages of false positive occurrence, thereby establishing a rapid detection system for large and small size insects, and providing a more efficient detection service.
首先簡述本發明之具體實施步驟,本發明係提供一種區分犬焦蟲症分型的方法,其步驟包括:(a)提供一核酸樣本;(b)對該核酸樣本進行一定量即時聚合酶鏈鎖反應(Q real-time PCR);以及(c)進行一熱熔解曲線(melting curve)分析以區分該核酸樣本之犬焦蟲症分型;其中該犬焦蟲症分型包括犬焦蟲(Babesia canis,BC)及吉布松焦蟲(Babesia gibsoni,Bg),且該即時定量聚合酶連鎖反應所使用之引子對SEQ ID NO:3及SEQ ID NO:4(BCBghsp231F/R)係針對該核酸樣本中編碼熱休克蛋白70基因之區段。較佳地,步驟(c)更包括進行一高解析熱熔解(High-Resolution Melting,HRM)分析;該核酸樣本係萃取自一個體之血液樣本;該血液樣本係為經過血球血漿分離後之血球樣本;其中步驟(C)更包括使用LC green染劑或SYBR green染劑。以上僅為簡述之實施步驟,其具體實施細節及實驗結果詳述如下。 First, the specific implementation steps of the present invention are briefly described. The present invention provides a method for distinguishing the classification of canine echinococcosis, the steps comprising: (a) providing a nucleic acid sample; and (b) performing a certain amount of immediate polymerase on the nucleic acid sample. a chain reaction (Q real-time PCR); and (c) performing a melting curve analysis to distinguish a canine worm typing of the nucleic acid sample; wherein the canine genus typing includes a canine worm ( Babesia canis , BC) and Babesia gibsoni (Bg), and the primer used in the real-time quantitative polymerase chain reaction is directed against SEQ ID NO: 3 and SEQ ID NO: 4 (BCBghsp231F/R) A segment of the heat shock protein 70 gene is encoded in the nucleic acid sample. Preferably, the step (c) further comprises performing a high-resolution melt-melting (HRM) analysis; the nucleic acid sample is extracted from a blood sample of the body; the blood sample is a blood cell separated by blood cell plasma. Sample; wherein step (C) further comprises using LC green dye or SYBR green dye. The above is only a brief implementation step, and the specific implementation details and experimental results are detailed below.
收集台灣各地動物醫院之臨床血液檢體共80個,以EDTA抗凝管低溫(4℃)保存運送至本公司進行檢體前處理(血漿血球分離)以利實驗進行,由於臨床檢體之變異性較大,可能見到貧血、溶血、脫水等狀況,故於核酸萃取前進行血漿血球分離,降低核酸萃取濃度上的差異,並提高檢體中所含之蟲體核酸濃度,避免偽陽性的發生。 A total of 80 clinical blood samples from animal hospitals in Taiwan were collected and stored in the EDTA anticoagulation tube at a low temperature (4 °C) for delivery to the company for pre-treatment (plasma hematocrit) for the purpose of experimentation. Sexually large, may see anemia, hemolysis, dehydration, etc., so plasma blood cell separation before nucleic acid extraction, reduce the difference in nucleic acid extraction concentration, and increase the concentration of worm nucleic acid contained in the sample to avoid false positive occur.
血漿血球分離方法(前處理)及全自動核酸萃取:將血漿血球進行3000rpm,15分鐘離心後取其下層血球200μL於96孔盤之第一及第七排,並依 序加入核酸萃取相關試劑於各反應排中(第一及七排加500μL lysis buffer,第二及第八排加50μL磁珠及750μL washing buffer A,第三及第九排加750μL washing buffer A,第四、五及第十、十一排加750μL washing buffer B,第六及十二排加50μL eluting buffer),套入磁棒套,按下start鍵後開始進行血球核酸萃取。 Plasma blood cell separation method (pretreatment) and automatic nucleic acid extraction: plasma blood cells are centrifuged at 3000 rpm for 15 minutes, and then 200 μL of the lower blood cells are taken in the first and seventh rows of the 96-well disk, and Add nucleic acid extraction related reagents to each reaction row (500 μL lysis buffer in the first and seventh rows, 50 μL magnetic beads and 750 μL washing buffer A in the second and eighth rows, and 750 μL washing buffer A in the third and ninth rows, Add 750μL washing buffer B to the fourth, fifth and tenth and eleventh rows, add 50μL eluting buffer to the sixth and twelve rows, insert the magnetic rod sleeve, and press the start button to start the blood cell nucleic acid extraction.
利用BC及Bg之熱休克蛋白(heat shock protein,hsp)70之高度差異性序列特性設計引子對BCBghsp231F/R(如圖13),編碼熱休克蛋白70之DNA片段分別是SEQ ID NO:1及SEQ ID NO:2,以DNAstar軟體分析該序列於BC及Bg上之GC content分別為55.41%及48.48%,預期其BC之熱溶解曲線溫度值大於Bg。其PCR反應試劑量分別為2X Taq預混料(premix)10μL、25mM MgCl2 1.2μL、DMSO 1μL、2uM BCBghsp231F/R 2μL、10uM template 5μL、DDW 0.8μL,以95℃,5分鐘之變性,95℃、58℃、72℃各30秒共35次循環,72℃,5分鐘之延長反應進行目標基因產物增幅反應,取PCR反應後之產物5μL加入SYBR green 5μL於384孔反應盤中,設定實驗模式為”standard curve”並包含PCR反應後之melt curve stage之反應條件為:95℃、15秒,60℃、1分鐘、95℃、15秒,以增溫速率每秒0.05℃偵測一次螢光值。 Using the highly differential sequence characteristics of BC and Bg heat shock protein (hsp) 70, the primer pair BCBghsp231F/R was designed (Fig. 13). The DNA fragment encoding heat shock protein 70 is SEQ ID NO: 1 and SEQ ID NO: 2, the GC content of the sequence on BC and Bg was 55.41% and 48.48%, respectively, and the thermal dissolution curve temperature value of BC was expected to be greater than Bg. The amount of PCR reaction reagent was 10 μL of 2X Taq premix, 1.2 μL of 25 mM MgCl 2 , 1 μL of DMSO, 2 μM of BCBghsp231F/R 2 μL, 10 μL of 10 μM template, 0.8 μL of DDW, denaturation at 95 ° C for 5 minutes, 95 At 35 ° C, 58 ° C, 72 ° C for 30 seconds, a total of 35 cycles, 72 ° C, 5 minutes to extend the reaction to the target gene product amplification reaction, take 5 μL of the product after PCR reaction, add SYBR green 5μL in a 384-well reaction plate, set the experiment The mode is "standard curve" and includes the reaction conditions of the melt curve stage after the PCR reaction: 95 ° C, 15 seconds, 60 ° C, 1 minute, 95 ° C, 15 seconds, detecting the firefly at a temperature increase rate of 0.05 ° C per second. Light value.
由於HR-1所需之螢光為LC green,可於PCR反應前或反應後外加,於成本考量上則以反應後經電泳確認有產物增幅後再外加LC green。另又因使用之2x Taq premix之PCR reagent為內含loading dye,為避免loading dye影響LC green嵌附於雙股DNA之效能,故另先行以其他不含loading dye 之Taq進行初篩,確認可行之Taq後再進行HRM。 Since the fluorescent light required for HR-1 is LC green, it can be added before or after the PCR reaction. In the cost consideration, after the reaction, it is confirmed by electrophoresis that the product is increased, and then LC green is added. In addition, the PCR reagent used in the 2x Taq premix is containing a loading dye. In order to avoid the influence of the loading dye on the binding of LC green to the double-stranded DNA, the other does not contain the loading dye. The Taq is screened and confirmed to be a viable Taq before HRM.
1. Taq測試:共進行三種Taq測試:(1)強力Taq(Power Taq)(2)熱啟動(Hot Start)(3)匹夫+(pfu plus),分別配製成每管反應體積為20μL之PCR預混料(premix):含有2.5mM MgCl2、0.2mM dNTP、0.2u/μL Taq之PCR premix備用。測試之檢體(DNA模板)共6支,分別為1.pBC(BC陽性對照)、2.pBg(Bg陽性對照)、3.檢體編號79之BC、4.Bg臨床檢體(120419002)、5.陰性臨床檢體、6.DDW,以2μL之引子對(2uM BCBghsp231F/R)及1μL之DNA模板。PCR條件:以95℃,5分鐘之變性,95℃、58℃、72℃各30秒共35次循環,72℃,5分鐘之延長反應進行目標基因產物增幅反應。電泳條件:以內含0.4mg/mL EtBr之2% agarose以250V之電流,15分鐘後於UV light下拍照並存檔。 1. Taq test: A total of three Taq tests were performed: (1) Power Taq (2) Hot Start (3) + (pfu plus), and each reaction volume was 20 μL per tube. PCR premix: PCR premix containing 2.5 mM MgCl 2 , 0.2 mM dNTP, 0.2 u/μL Taq. A total of 6 samples (DNA template) were tested, 1. pBC (BC positive control), 2. pBg (Bg positive control), 3. BC with sample number 79, 4. Bg clinical sample (120419002) 5. Negative clinical specimen, 6.DDW, 2 μL primer pair (2uM BCBghsp231F/R) and 1 μL DNA template. PCR conditions: a denaturation reaction at 95 ° C, 5 minutes, 95 ° C, 58 ° C, 72 ° C for 30 seconds for a total of 35 cycles, 72 ° C, 5 minutes extended reaction to the target gene product amplification reaction. Electrophoresis conditions: 2% agarose containing 0.4 mg/mL EtBr at 250 V, 15 minutes later photographed under UV light and archived.
2. 以LC green進行HR-1分析:測試之BC、Bg檢體(DNA模板)各5支,分別為BC:1.pBC(BC陽性對照)、2.檢體編號79、3.檢體編號40、4.未編檢體AM111008006及5.檢體編號57;Bg:6.pBg(Bg陽性對照)、7.未編檢體AM120419002、8.檢體編號10、9.檢體編號23、10.檢體編號29,11.陰性檢體、12.DDW。引子對:2uM BCBghsp231F/R。PCR premix同上述以最終體積為20μL分別加入1μL之DNA模板及2μL引子對。PCR條件及跑膠條件同上述。HRM:將經電泳確認有產物增幅之檢體取9μL加1μL LC green以pipette充分混合後置於玻璃毛細管(Light Cycler,USA)並短暫離心,將待測產物置入分析儀中,所設條件為:升溫速度0.3℃/秒、始測螢光溫度60℃、終止溫度98℃。 2. HR-1 analysis by LC green: 5 BC and Bg samples (DNA template), respectively, BC: 1.pBC (BC positive control), 2. Sample number 79, 3. Sample No. 40, 4. Unedited sample AM111008006 and 5. Sample number 57; Bg: 6.pBg (Bg positive control), 7. Unedited sample AM120419002, 8. Sample number 10, 9. Sample number 23 10. Sample No. 29, 11. Negative specimen, 12.DDW. Primer pair: 2uM BCBghsp231F/R. The PCR premix was added to 1 μL of the DNA template and 2 μL of the primer pair in the final volume of 20 μL as described above. The PCR conditions and running conditions are the same as above. HRM: 9 μL of the sample confirmed by electrophoresis and 1 μL of LC green was thoroughly mixed with a pipette, placed in a glass capillary (Light Cycler, USA) and centrifuged briefly, and the product to be tested was placed in the analyzer. The temperature is 0.3 ° C / sec, the initial fluorescence temperature is 60 ° C, and the termination temperature is 98 ° C.
3. 確定可行後,再以上述之方法進行BC及Bg檢體(各16支,表4)之HR-1分析。 3. After the determination is feasible, perform HR-1 analysis of BC and Bg samples (16 each, Table 4) by the above method.
4. 測試SYBR green於HR-1分析:所測試之檢體同2.,但其為2x之premix,故取10μL之2x SYBR green premix,加入1μL之DNA模板、2μL引子對及DDW 7μL,PCR、跑膠條件及HRM方法同上述。 4. Test SYBR green in HR-1 analysis: the tested sample is the same as 2., but it is 2x premix, so take 10μL of 2x SYBR green premix, add 1μL DNA template, 2μL primer pair and DDW 7μL, PCR The running conditions and the HRM method are the same as above.
以具有多個鹼基差異的BCBghsp231F/R引子對進行測試。BC之測試 檢體為10μg/μL pBC+(BC陽性對照)、檢體編號47、59、63、64、66、67;Bg之測試檢體為10μg/μL pBg+(Bg陽性對照)、檢體編號56、54、53、51、50、49,其中pBC及pBg則另以1:1混合後加入測試。所使用之試劑為2x Melt DoctorTM HRM reagent,配製成每管反應體積為10μL之PCR premix:5μL之2x Melt DoctorTM HRM reagent(含SYTO 9染劑)、2μL之2uM引子對、1μL之測試檢體、2μL之DDW。於384孔反應盤中分別標記檢體位置並以光學反應膜服貼於其上後短暫離心(3000rpm,1分鐘),放入ViiATM7分析儀中進行檢體分析,設定實驗模式為”High Resolution Melting”並包含PCR反應,其反應條件為:Hold stage:95℃,10分鐘;PCR stage(40次循環(cycles)):95℃、15秒,60℃、1分鐘;HRM stage:95℃、10秒,60℃、1分鐘,95℃、15秒(每秒0.025℃偵測一次螢光值)。 The BCBghsp231F/R primer pair with multiple base differences was tested. The test specimens of BC were 10 μg/μL pBC+ (BC positive control), sample numbers 47, 59, 63, 64, 66, 67; Bg test samples were 10 μg/μL pBg+ (Bg positive control), sample number 56, 54, 53, 51, 50, 49, wherein pBC and pBg were mixed with 1:1 and added to the test. The reagents used for the 2x Melt Doctor TM HRM reagent, formulated in a reaction volume of 10μL per tube of PCR premix: 5μL of 2x Melt Doctor TM HRM reagent (containing dye SYTO 9), 2uM 2μL of pair of primers, 1μL of test Sample, 2 μL of DDW. In 384-well plate positions are marked subject and obedient to the optical reaction film thereon after brief centrifugation (3000rpm, 1 min), placed ViiA TM 7 analyzer for analyzing a specimen, is set as the experimental model "High Resolution Melting" and includes a PCR reaction under the following conditions: Hold stage: 95 ° C, 10 minutes; PCR stage (40 cycles): 95 ° C, 15 seconds, 60 ° C, 1 minute; HRM stage: 95 ° C , 10 seconds, 60 ° C, 1 minute, 95 ° C, 15 seconds (0.025 ° C per second to detect a fluorescent value).
另外為進行蟲體濃度之定量,使用已知之pBC及pBg濃度(10ug/uL)以1:1混合進行對半序列稀釋(seim-dilute)(圖31-1,將所得之ct值套入回歸公式進而得到未知檢體之濃度。 In addition, in order to quantify the concentration of the worm, a semi-sequence dilution (seim-dilute) was performed using a known 1:1 mixture of pBC and pBg (10 ug/uL) (Fig. 31-1, the obtained ct value was placed in the regression). The formula then obtains the concentration of the unknown sample.
收集台灣各地動物醫院臨床血液檢體,以EDTA抗凝管低溫(4℃)保存運送至本公司進行檢體前處理。由於臨床檢體呈現多樣性(貧血、溶血、脫水等)常造成檢體核酸萃取後之差異性提高以致影響檢測之準確度,於是利用焦蟲寄生於宿主紅血球內的特性,採用血漿血球分離方式提高蟲體核酸濃度以利實驗進行,如圖2。 The clinical blood samples of animal hospitals in Taiwan were collected and stored in the EDTA anticoagulation tube at a low temperature (4 °C) and transported to the company for pre-treatment. Due to the diversity of clinical samples (anemia, hemolysis, dehydration, etc.), the difference in the nucleic acid extraction of the sample often increases the accuracy of the detection, so the characteristics of the parasites in the host red blood cells are utilized, and the blood cell separation method is adopted. Increasing the nucleic acid concentration of the worm to facilitate the experiment, as shown in Figure 2.
目前已收集80個臨床檢體,所得之平均濃度為57.14ng/μL,比值為1.78,此80個臨床檢體中可見其濃度值亦有所差異,推測為檢體本身原因,濃度頗高者可能為受感染檢體(白血球、噬中性球、單核球、淋巴球增加)、再生性貧血檢體(網織球增加),濃度低者可能由於非再生性貧血、溶血檢體等。 At present, 80 clinical samples have been collected, and the average concentration is 57.14 ng/μL, the ratio is 1.78. The concentration values of the 80 clinical samples are also different, which is presumed to be the cause of the sample itself. It may be infected specimens (white blood cells, neutrophils, mononuclear spheres, lymphocytes), regenerative anemia specimens (increased reticular balls), and low concentrations may be due to non-regenerative anemia, hemolysis specimens, etc.
在此所述的檢體收集係採用血球與血漿分離之方式,血液主要由紅血 球和血漿組成,分別占血液的40%和55%。由於焦蟲會寄生在紅血球中,因此將檢體中的血球與血漿分離可以減去血漿中核酸的干擾,提升焦蟲核酸的濃度,降低因為焦蟲核酸的濃度太稀造成檢驗上的誤差。另外,核酸萃取係利用磁珠好吸附與安定的特性,在磁珠上修飾官能基團,利用官能基團與核酸的吸附作用達到從檢體中快速取得核酸與分離的效果。全程自動化的設計不僅節省時間成本、對於取得核酸的量與純度上也大有改善。 The sample collection system described herein uses a method in which blood cells are separated from plasma, and blood is mainly composed of red blood. The ball and plasma make up 40% and 55% of the blood, respectively. Since the coccidia will be parasitic in the red blood cells, separating the blood cells from the plasma in the sample can reduce the interference of nucleic acid in the plasma, increase the concentration of the coccidial nucleic acid, and reduce the error in the test due to the too small concentration of the coccidio nucleic acid. In addition, the nucleic acid extraction utilizes the characteristics of good adsorption and stability of the magnetic beads, modifying the functional groups on the magnetic beads, and utilizing the adsorption of the functional groups and the nucleic acids to achieve the effect of rapidly obtaining nucleic acid and separation from the sample. The entire automated design not only saves time and costs, but also greatly improves the amount and purity of nucleic acids.
本實驗利用大小焦蟲heat shock protein 70序列之高差異度特性設計該序列核試之引子對(BCBghsp231F/R)進行PCR,確認增幅出大小焦蟲之heat shock protein 70產物(如圖14),再將其產物進行熱溶解曲線測試結果(如圖15),其熱溶解曲線溫度值為BC>Bg(表2)。 In this experiment, the primers of the sequence nuclear test (BCBghsp231F/R) were designed by PCR using the high-difference characteristics of the heat shock protein 70 sequence, and the heat shock protein 70 product of the size-cavity was confirmed (Fig. 14). The product was then subjected to a thermal dissolution curve test result (Fig. 15), and the thermal dissolution curve temperature value was BC > Bg (Table 2).
1. Taq測試結果以Power Taq效果最佳(如圖16)。 1. Taq test results are best with Power Taq (Figure 16).
2. 測試之BC、Bg檢體以LC green進行HR-1分析前進行PCR之結果如圖17,將有增幅出產物之檢體進行HR-1之結果如圖18,可初步得知其BC之Tm值大於Bg。 2. The results of the PCR performed before the HR-1 analysis of the BC and Bg samples tested by LC green are shown in Fig. 17. The results of the HR-1 of the sample with the amplified product are shown in Fig. 18, and the BC can be initially known. The Tm value is greater than Bg.
3. 經上述1.及2.之測試結果可行後,進一步以BC及Bg臨床檢體以LC green進行HR-1測試(如圖19、20)。 3. After the test results of 1. and 2. above are feasible, the HR-1 test is further performed with LC green in BC and Bg clinical samples (Figures 19 and 20).
4. 測試SYBR green於HR-1分析之結果(如圖21、22)。 4. Test the results of SYBR green in HR-1 analysis (Figures 21, 22).
本實驗測試之BC及Bg檢體heat shock protein 70基因之Ct值與Tm值如表5,於heat shock protein 70測試之結果:擴增曲線圖(amplification plot)如圖25、26,Raw melt curve如圖27,導函數熔解曲線(derivative melt curve) 結果如圖28,信號歸一化(aligned melt curves)結果如圖29及差異曲線圖(difference plot)如圖30。 The Ct and Tm values of the BC and Bg samples of the heat shock protein 70 gene tested in this experiment are shown in Table 5. The results of the heat shock protein 70 test: the amplification plot is shown in Figures 25 and 26, Raw melt curve. As shown in Figure 27, the derivative melt curve (derivative melt curve) The results are shown in Fig. 28. The results of the aligned melt curves are shown in Fig. 29 and the difference plot is shown in Fig. 30.
定量結果如圖31-2及表5,各大小焦蟲陽性檢體ct值各為6.24、10.66、14.52、19.23、22.97、26.55、29.62,測試之大焦蟲陽性檢體(BC+)ct值為17.85,藉由對半稀釋之已知濃度(0.5、0.25、0.125、0.0625、0.03125、0.015625、0.0078125ug/uL)取得回歸曲線公式(y=5.686ln(x)+2.7759),套入公式後得測試之大焦蟲陽性檢體濃度為0.0003191ug/uL;分型結果如圖32,對照陽性檢體Tm值可確認測試檢體為大焦蟲。 Quantitative results are shown in Figure 31-2 and Table 5. The ct values of the positive samples of each size were 6.24, 10.66, 14.52, 19.23, 22.97, 26.55, 29.62. The CCD value of the large coccidia positive test (BC+) was tested. 17.85, by using the known concentration of half-dilution (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125 ug / uL) to obtain the regression curve formula (y = 5.686ln (x) + 2.7759), after the formula The concentration of the positive cokeworm positive sample tested was 0.00031191 ug/uL; the typing result is shown in Fig. 32. The Tm value of the positive test sample can confirm that the test sample is a large coke.
經由上述所揭露之具體實施方法及所得結果,可得到以下討論。上述實施例所採用之檢體核酸萃取方式,係採血漿血球分離方式,其可增加蟲體濃度、避免貧血、溶血或蟲體含量較低所造成的假陰性結果。 The following discussion can be obtained by the specific implementation methods disclosed above and the results obtained. The sample nucleic acid extraction method used in the above embodiments is a plasma blood cell separation method, which can increase the concentration of the insect body, avoid false negative results caused by anemia, hemolysis or low content of the worm.
關於以heat shock protein 70進行BC及Bg區分之結果,由於習知鑑定方式所花費時間較長,另以BC及Bg之heat shock protein 70進行區分,利用兩種蟲體之heat shock protein 70具高度差異性之特性設計適合之引子對,進行產物增幅後加入SYBR green以ViiA7TM中"melt curve"實驗模式進行分析,其結果可成功地將BC及Bg以Tm值區分開來,但其所呈現之熔解曲線圖較為粗糙(與實際以HRM測試結果有一定程度之落差)。相較於以heat shock protein 70直接以ViiA7TM(Applied Biosystems,USA)中"standard curve"實驗模式進行BC及Bg檢體Tm值分析方式可行性高,所測試之LC green及SYBR green兩種螢光之結果應用於ViiA7TM及需先將產物進行增幅之HR-1(Idaho Technology)兩儀器中顯示:LC green於HR-1測試結果較SYBR green佳(所得之熱熔解曲線較細緻),亦可於PCR反應前或反應後外加螢光,可先行以電泳確認產物增幅後再行外加螢光,因而可省去耗材費用,而LC green則是已加入酵素之2x PCR premix,故較浪費酵素用量;以操作上而言,HR-1僅一次上一個檢體(玻璃毛細管),且須操作人員在側,於檢體分析後再以人為操作方式進行下一檢體之分析,所花費之人力較大,而於ViiA7TM操作上,其為384 well反應盤,雖不及玻璃毛細管來的佳,但高通量之優勢足以為勝,且該儀器亦有擴增曲線結果可得Ct值,進一步換算蟲體量(濃度),亦於HRM分析軟體中涵括熔解曲線之Raw data、導函數圖、螢光信號歸一化及差異圖(difference plot)等強大之功能,且所使用之螢光(SYTO 9)為原廠附之含之HRM專用試劑(Melt Dr,HRM reagent),為2x premix,過程中不需像HR-1要以電泳方式進行產物確認,直接上機即可得擴增曲線圖。 Regarding the results of BC and Bg discrimination by heat shock protein 70, because of the long time taken by the conventional identification method, the heat shock protein 70 of BC and Bg is distinguished, and the heat shock protein 70 height of the two insect bodies is utilized. The characteristics of the difference are designed to be suitable for the primer pair. After the product is amplified, SYBR green is added to analyze the "melt curve" experimental mode in ViiA7 TM . The result can successfully distinguish BC and Bg by Tm value, but it is presented. The melting curve is rougher (there is a certain degree of difference with the actual HRM test results). Compared with the heat shock protein 70 directly in the "standard curve" experimental mode of ViiA7 TM (Applied Biosystems, USA), the method of analyzing the Tm value of BC and Bg samples is highly feasible, and the LC green and SYBR green tested are high. The results of the light were applied to the ViiA7 TM and the HR-1 (Idaho Technology) instrument which required the first increase of the product: LC green was better than the SYBR green in the HR-1 test (the obtained thermal melting curve was finer) Fluorescence can be added before or after the PCR reaction. It can be confirmed by electrophoresis to increase the product and then add fluorescence. This saves the cost of consumables. LC green is a 2x PCR premix that has been added to the enzyme, so it is a waste of enzyme. In terms of operation, HR-1 is only used once on the first specimen (glass capillary), and the operator must be on the side. After the analysis of the specimen, the next specimen is analyzed by human operation. The manpower is large, and on the ViiA7 TM operation, it is a 384 well reaction plate. Although it is better than the glass capillary, the advantage of high throughput is enough, and the instrument also has the amplification curve result to obtain the Ct value. Further conversion of the amount of insects (concentrated In the HRM analysis software, the Raw data, the derivative function diagram, the fluorescence signal normalization, and the difference plot including the melting curve are powerful functions, and the fluorescent light (SYTO 9) used is the original. The HRM reagent (Melt Dr, HRM reagent) included in the factory is 2x premix. During the process, it is not necessary to confirm the product by electrophoresis like HR-1, and the amplification curve can be obtained directly on the machine.
另外,關於以兩種螢光/核酸染劑(LC green及SYBR green)應用於HR-1儀器之結果而言:1.兩種螢光應用於BCBghsp引子對皆可成功增幅出目標產物,並需使用HR-1專用之玻璃毛細管進行檢體分析。2.兩者皆需經由 電泳確認後才可進行HR-1分析:於LC green,其可於PCR反應後外加,較省試劑量,但LC green為單純螢光,需另外製備PCR premix;於SYBR green,其為2x premix,不需額外製備PCR premix,但其缺點則由於內已含SYBR green螢光,無法由電泳確認後再行添加,較為浪費。3.於HR-1分析之結果:1.其Tm值(熔解曲線溫度)於BC及Bg之差相似(LC green為2.3℃,SYBR green為2.34℃)。2.其Tm值於LC green之值大於SYBR green之值(LC green之BC為89.73℃,Bg為87.43℃;LC green之BC為87.56℃,Bg為85.22℃),由其結果可推測SYBR green較無法偵測到異源雙股結構(heteroduplex),而LC green不僅可偵測異源雙股結構,其所能觀察到的熔解曲線溫度變化比SYBR green更寬廣(更細緻),Liew等人更指出LC green可用於快速偵測同源合子(homogeneous)中之異源雙股結構且不會抑制PCR產物之增幅,亦不需特殊探針或試劑,只需特定引子對、一次PCR與分析儀。以LC green於HRM之應用上強調其可偵測出單一鹼基之差異,此方式常應用於單一點突變、抗藥性基因偵測。 In addition, regarding the results of applying two kinds of fluorescent/nucleic acid dyes (LC green and SYBR green) to the HR-1 instrument: 1. Both kinds of fluorescence applied to the BCBghsp primer pair can successfully increase the target product, and Sample analysis is required using a glass capillary for HR-1. 2. Both need to go through HR-1 analysis can be performed after electrophoresis confirmation: in LC green, it can be added after the PCR reaction, which is more than the reagent amount, but LC green is pure fluorescence, and additional PCR premix is needed; in SYBR green, it is 2x premix There is no need to prepare additional PCR premix, but its shortcomings are because it contains SYBR green fluorescence, which cannot be confirmed by electrophoresis and is added. 3. Results of HR-1 analysis: 1. The Tm value (melting curve temperature) is similar between BC and Bg (LC green is 2.3 ° C, SYBR green is 2.34 ° C). 2. The value of Tm value in LC green is greater than the value of SYBR green (LC green BC is 89.73 ° C, Bg is 87.43 ° C; LC green BC is 87.56 ° C, Bg is 85.22 ° C), from which the results can be inferred SYBR green It is less able to detect the heteroduplex structure, and LC green can not only detect the heterogeneous double-strand structure, but the melting curve temperature can be observed to be wider (more detailed) than SYBR green, Liew et al. It is further pointed out that LC green can be used to rapidly detect heterologous double-stranded structures in homologous homologues without inhibiting the amplification of PCR products, without special probes or reagents, only specific primer pairs, one PCR and analysis. instrument. The application of LC green to HRM emphasizes that it can detect the difference of single base, which is often applied to single point mutation and drug resistance gene detection.
本實驗為分析BC及Bg本身之heat shock protein 70,利用其多點之鹼基差異,加入螢光並使其鑲嵌於雙股核酸上,再利用HRM原理進行螢光降解率的分析以求得目標基因之熔解曲線溫度,藉由BC及Bg之熔解曲線溫度差異求得區分之目的,且本實驗之最終目的並非偵測單一鹼基之差異,故於螢光物質之使用上並無太多限制。另外,為求達到定量目的,以HRM分析儀(HR-1,Idaho technology)僅能偵測螢光強度及Tm值,無法達到定量效果,而以ViiA7儀中之HRM實驗模式不但可得其Tm值,亦可以其PCR系統中求得Ct值並進一步換算未知蟲體之核酸濃度。 This experiment is to analyze the heat shock protein 70 of BC and Bg itself, using its multiple base differences, adding fluorescence and embedding it on the double-stranded nucleic acid, and then using the HRM principle to analyze the fluorescence degradation rate. Melting curve temperature of target gene, melting curve by BC and Bg Temperature difference The purpose of the distinction is sought, and the ultimate goal of the experiment is not to detect differences in single bases, so there is not much restriction on the use of fluorescent substances. In addition, in order to achieve quantitative purposes, the HRM analyzer (HR-1, Idaho technology) can only detect the fluorescence intensity and Tm value, and the quantitative effect cannot be achieved. However, the HRM experimental mode in the ViiA7 instrument can not only obtain the Tm. The value can also be used to determine the Ct value in the PCR system and further convert the nucleic acid concentration of the unknown worm.
BC及Bg之定量:本實驗針對BC及Bg之18S rDNA所設計之探針並未能達區分目的,因而使用heat shock protein 70進行HRM測試,其結果可行,可於ViiA7儀中之HRM實驗模式成功地將BC及Bg區分開來,可見原始熔解曲線(Raw melt curve)、導函數熔解曲線(derivative melt curve)結果、信號歸一化(aligned melt curves)之結果,另外更可於擴增曲線圖(amplification plot)中得到待測檢體之Ct值,利用已知濃度之陽性對照組及其Ct值進行待測檢體之絕對定量換算。 Quantification of BC and Bg: The probe designed for 18S rDNA of BC and Bg did not reach the purpose of differentiation. Therefore, heat shock protein 70 was used for HRM test. The result is feasible and can be used in the HRM experimental mode of ViiA7. Successfully distinguishing between BC and Bg, the results of the Raw melt curve, the derivative melt curve, the aligned melt curves, and the amplification curve. The Ct value of the sample to be tested is obtained in an amplification plot, and the absolute quantitative conversion of the sample to be tested is performed using the positive control group of known concentration and its Ct value.
綜上所述,以犬焦蟲與吉布松焦蟲之heat shock protein 70進行高解析熱熔解分析(high resolution melting)方式可成功地將兩種蟲體進行區分,並利用已知濃度陽性對照組之Ct值求得待測蟲體濃度(可達絕對定量),此檢測方式約四小時(包含核酸萃取及HRM上機時間),已屬相當快速之實驗方式,大大提供了臨床獸醫師所需之治療黃金時間。在未來可利用本發明所揭露之技術內容,其具有提升檢測精確度、降低偽陽性發生之優點,根據該些技術內容可建立區分犬大小焦蟲的快速檢測系統,提供更有效率的檢測服務。 In summary, the high resolution melting method of the heat shock protein 70 of canine worm and Gibbs pine can successfully distinguish the two worms and use the known concentration positive control. The Ct value of the group is obtained to determine the concentration of the tested worm (up to an absolute quantitative value). The detection method is about four hours (including nucleic acid extraction and HRM on-board time), which is a very fast experimental method and greatly provides a clinical veterinarian clinic. Need to treat prime time. In the future, the technical content disclosed by the present invention can be utilized, which has the advantages of improving detection accuracy and reducing the occurrence of false positives. According to the technical contents, a rapid detection system for distinguishing canine size insects can be established, and a more efficient detection service can be provided. .
由以上實施例可知,本發明所提供之利用基因檢測分析性格的方法確具產業上之利用價值,惟以上之敘述僅為本發明之較佳實施例說明,凡精於此項技藝者當可依據上述之說明而作其它種種之改良,惟這些改變仍屬於本發明之精神及以下所界定之專利範圍中。 It can be seen from the above embodiments that the method for utilizing the gene detection and analysis character provided by the present invention has industrial utilization value, but the above description is only for explaining the preferred embodiment of the present invention, and those skilled in the art can Other various modifications are possible in light of the above teachings, but such changes are still within the spirit of the invention and the scope of the invention as defined below.
1. Birkenheuer, A. J., Levy, M. G., Breitschwerdt, E. B. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. Journal of Clinical Microbiology. 41(9): 4172~4177, 2003. 1. Birkenheuer, AJ, Levy, MG, Breitschwerdt, EB Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. Journal of Clinical Microbiology. 41(9) : 4172 ~ 4177, 2003.
2. Lee, C. C., Huang, C. C., Tsang, C. L., Chung, Y. T. Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan. Journal ofveterinary medicine science. 72(10): 1329~1335, 2010. 2. Lee, CC, Huang, CC, Tsang, CL, Chung, YT Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein (TRAP) gene of Babesia gibsoni isolates from dogs in Taiwan. Journal of veterinary medicine science. 72(10) : 1329 ~ 1335, 2010.
3. Matjila, P. T., Leisewitz, A. L., Jongejan, F., Bertschinger, H. J., PenZhorn, B. L. Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Afica. Veterinary Parasitology. 157: 123~127,2008. 3. Matjila, PT, Leisewitz, AL, Jongejan, F., Bertschinger, HJ, PenZhorn, BL Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Afica. Veterinary Parasitology. 157: 123 ~ 127, 2008.
4. Zahler, M., Schein, E., Rinder, H., Gothe, R. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs. Parasitology Research. 84: 544~548,1998. 4. Zahler, M., Schein, E., Rinder, H., Gothe, R. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs. Parasitology Research. 84: 544 ~ 548, 1998.
5. Wittwer, C. T., Reed, G. H., Gundry, C. N., Vandersteen, J. G., Pryor, R. J. High-resolution genotyping by amplicon melting analysis using LCGreen. Clinical Chemistry. 49(6): 853~860, 2003. 5. Wittwer, CT, Reed, GH, Gundry, CN, Vandersteen, JG, Pryor, RJ High-resolution genotyping by amplicon melting analysis using LC Green. Clinical Chemistry. 49(6): 853 ~ 860, 2003.
6. Liew, M., Pryor, R., Palais, R., Meadows, C., Erali, M., Lyon, E., Wittwer, C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clinical Chemistry. 50(7): 1156~1164, 2004. 6. Liew, M., Pryor, R., Palais, R., Meadows, C., Erali, M., Lyon, E., Wittwer, C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons . Clinical Chemistry. 50(7): 1156 ~ 1164, 2004.
圖1為犬焦蟲(Babesia canis,BC)及吉布松焦蟲(Babesia gibsoni,Bg)18S rDNA序列比對分析結果的示意圖。 Figure 1 is a schematic diagram showing the results of 18S rDNA sequence alignment analysis of Babesia canis (BC) and Babesia gibsoni (Bg).
圖2為臨床血液檢體採取其血球層(RBC/buffy coat)進行核酸萃取的示意圖。 2 is a schematic diagram of a nucleic acid extraction by a clinical blood sample using its blood cell layer (RBC/buffy coat).
圖3為犬焦蟲(Babesia canis,BC)及吉布松焦蟲(Babesia gibsoni,Bg)18S rDNA引子對(BCBg308F/R)的示意圖。 Figure 3 is a schematic diagram of the 18S rDNA primer pair (BCBg308F/R) of Babesia canis (BC) and Babesia gibsoni (Bg).
圖4為BC與Bg共同之18S rDNA引子對BCBg308F/R進行臨床檢體聚合酶鏈鎖反應後電泳結果的示意圖。 Fig. 4 is a schematic diagram showing the results of electrophoresis of a BCBg308F/R by a 18S rDNA primer combined with BC and Bg.
圖5為Bg之TRAP序列設計nested PCR之引子對共兩組(綠框為1st PCR引子對,藍框為2nd PCR引子對)的示意圖。 Fig. 5 is a schematic diagram showing the introduction of nested PCR primers for the TLP sequence of Bg (the green frame is a 1st PCR primer pair and the blue frame is a 2nd PCR primer pair).
圖6為BgTRAP1091引子對進行nested PCR之一次PCR結果的示意圖。 Figure 6 is a schematic diagram showing the results of one PCR performed on the nested PCR of the BgTRAP1091 primer pair.
圖7為BgTRAP481引子對進行nested PCR之二次PCR結果的示意圖。 Figure 7 is a schematic diagram showing the results of secondary PCR of nested PCR of BgTRAP481 primer pair.
圖8為以引子對BCBg308F/R設計之BC及Bg探針(藍色框為BC probe1、紅色框為Bg probe2)的示意圖。 Figure 8 is a schematic diagram of the BC and Bg probes designed with the primer pair BCBg308F/R (blue frame for BC probe1 and red for Bg probe2).
圖9為以ViiA7”standard curve”實驗模式進行BC及Bg檢體之BC probe 1測試之擴增曲線圖(amplification plot)的示意圖。 Figure 9 is a schematic diagram of an amplification plot of the BC probe 1 test of BC and Bg samples in the ViiA7 "standard curve" experimental mode.
圖10為以ViiA7”standard curve”實驗模式進行BC及Bg檢體之BC probe 2測試之擴增曲線圖(amplification plot)的示意圖。 Figure 10 is a schematic diagram of an amplification plot of the BC probe 2 test of BC and Bg samples in the ViiA7 "standard curve" experimental mode.
圖11為以Mfe I限制酶切作用位置(必於藍框處作用於BC及Bg,於紅框處僅作用於Bg)的示意圖。 Figure 11 is a schematic diagram showing the position of restriction enzyme digestion by Mfe I (which must act on BC and Bg at the blue frame and only Bg at the red frame).
圖12為BC、Bg檢體以BC、Bg共同引子對BCBg308F/R進行PCR增幅後之產物利用Mfe I進行限制酶切結果的示意圖。 Fig. 12 is a schematic diagram showing the results of restriction enzyme digestion of products of BC and Bg samples by PCR amplification of BCBg308F/R by BC and Bg co-introduction.
圖13為BC、Bg之heat shock protein 70序列之引子對位置(綠色框),其中藍色框可見BC及Bg之差異處的示意圖。 Figure 13 is a schematic representation of the position of the primer pair (green box) of the heat shock protein 70 sequence of BC and Bg, wherein the blue box shows a schematic representation of the difference between BC and Bg.
圖14為BC、Bg檢體以heat shock protein 70進行PCR結果的示意圖。 Fig. 14 is a schematic diagram showing the results of PCR performed by heat shock protein 70 on BC and Bg samples.
圖15為BC、Bg檢體以heat shock protein 70之PCR增幅產物進行熱溶解曲線分析結果的示意圖。 Fig. 15 is a view showing the results of thermal dissolution curve analysis of BC and Bg samples by PCR amplification products of heat shock protein 70.
圖16為Taq測試結果的示意圖。 Figure 16 is a schematic diagram of the Taq test results.
圖17為HR-1分析前進行PCR確認結果的示意圖。 Figure 17 is a schematic diagram showing the results of PCR confirmation before HR-1 analysis.
圖18為將圖17之PCR產物進行HR-1分析後之導函數(derivative plot)結果的示意圖。 Figure 18 is a graph showing the results of a derivative plot of the PCR product of Figure 17 after HR-1 analysis.
圖19為BC、Bg檢體於HR-1分析前進行PCR確認結果(未加LCgreen) Figure 19 shows the results of PCR confirmation of BC and Bg samples before HR-1 analysis (without LCgreen)
圖20為BC、Bg之heat shock protein 70以HR-1進行HRM之導函數(derivative plot)結果(圖19產物+LCgreen)的示意圖。 Figure 20 is a schematic diagram showing the results of the HRM derivative plot of BC, Bg heat shock protein 70 (Fig. 19 product + LCgreen).
圖21為BC、Bg檢體於HR-1分析前進行PCR確認結果(SYBR green premix)的示意圖。 Fig. 21 is a schematic diagram showing the results of PCR confirmation (SYBR green premix) of BC and Bg samples before HR-1 analysis.
圖22為BC、Bg之heat shock protein 70以HR-1進行HRM之導函數(derivative plot)結果(圖21產物+LCgreen)的示意圖。 Fig. 22 is a schematic diagram showing the results of the HRM derivative plot of BC, Bg heat shock protein 70 (Fig. 21 product + LCgreen).
圖23為以BCBgprobe2引子對進行BC、Bg檢體之18S rDNA增幅結果(於HR-1分析前進行PCR確認結果)的示意圖。 Fig. 23 is a schematic diagram showing the results of 18S rDNA amplification of BC and Bg samples by BCBgprobe2 primer pair (the results of PCR confirmation before HR-1 analysis).
圖24為BC、Bg之18S rDNA以HR-1進行HRM之導函數(derivative plot)結果(圖23產物+LCgreen)的示意圖。 Fig. 24 is a schematic diagram showing the results of the derivative plot of HRM of BC, Bg 18S rDNA with HR-1 (Fig. 23 product + LCgreen).
圖25為以ViiA7之”High Resoultion Melting”實驗模式進行BC之heat shock protein 70擴增曲線圖(amplification plot)的示意圖。 Figure 25 is a schematic illustration of the BC heat shock protein 70 amplification plot in ViiA7 "High Resoultion Melting" experimental mode.
圖26為以ViiA7之”High Resoultion Melting”(HRM)實驗模式進行Bg之heat shock protein 70擴增曲線圖(amplification plot)的示意圖。 Figure 26 is a schematic diagram showing the heat shock protein 70 amplification plot of Bg in the "High Resoultion Melting" (HRM) experimental mode of ViiA7.
圖27為BC、Bg及BCBg mixed之heat shock protein 70以ViiA7進行HRM之結果(Raw melt curve)的示意圖。 Figure 27 is a graphical representation of the Raw melt curve for BC, Bg, and BCBg mixed heat shock protein 70 with ViiA7.
圖28為BC、Bg及BCBg mixed之heat shock protein 70以ViiA7進行HRM之導函數熔解曲線(derivative melt curve)結果的示意圖。 Figure 28 is a graph showing the results of a derivative melt curve of HRM by VII, Bg and BCBg mixed heat shock protein 70 with ViiA7.
圖29為BC、Bg及BCBg mixed之heat shock protein 70以ViiA7進行HRM之信號歸一化後之結果的示意圖。 Fig. 29 is a graph showing the results of normalization of HRM signals by ViiA7 using BC, Bg and BCBg mixed heat shock protein 70.
圖30為BC、Bg及BCBg mixed之heat shock protein 70以ViiA7進行 HRM之差異曲線圖(difference plot)的示意圖。 Figure 30 shows BC, Bg and BCBg mixed heat shock protein 70 with ViiA7 Schematic diagram of the difference plot of HRM.
圖31-1為利用已知之pBC及pBg濃度(10ug/uL)以1:1混合進行對半序列稀釋(semi-dilute)的示意圖。 Figure 31-1 is a schematic diagram of semi-dilute dilution using a known concentration of pBC and pBg (10 ug/uL) in a 1:1 mix.
圖31-2為使用已知之pBC及pBg濃度(10ug/uL)以1:1混合進行對半序列稀釋(seim-dilute),以ViiA7進行HRM之差異曲線圖(difference plot),BC+為已知之陽性檢體的示意圖。 Figure 31-2 shows the difference between the semi-sequence dilution (seim-dilute) using a known 1:1 pBC and pBg concentration (10 ug/uL) and the HRM with ViiA7. BC+ is known. A schematic representation of a positive specimen.
圖32為使用已知之pBC及pBg濃度(10ug/uL)以1:1混合進行對半序列稀釋(seim-dilute),以ViiA7進行HRM之導函數熔解曲線(derivative melt curve),BC+為已知之陽性檢體的示意圖。 Figure 32 is a derivative melt curve for the HRM with ViiA7 using a known 1:1 mixture of pBC and pBg concentrations (10 ug/uL) in a 1:1 mixture, BC+ is known. A schematic representation of a positive specimen.
圖33為犬焦蟲(Babesia canis)定序結果及NCBI blast結果的示意圖。 Figure 33 is a schematic diagram showing the results of the sequence of Babesia canis and NCBI blast results.
圖34為吉布松焦蟲(Babesia gibsoni)定序結果及NCBI blast結果的示意圖。 Figure 34 is a schematic diagram showing the results of sequencing of Babesia gibsoni and NCBI blast results.
圖35為吉布松焦蟲(Babesia gibsoni)TRAP gene定序結果及NCBI blast結果的示意圖。 FIG 35 is a Jibu Song Babesia (Babesia gibsoni) sequencing schematic NCBI blast results and results TRAP gene.
圖36為犬焦蟲(Babesia canis)heat shock protein 70定序結果及NCBI blast結果。 Figure 36 shows the results of sequencing of Babesia canis heat shock protein 70 and NCBI blast results.
圖37為吉布松焦蟲(Babesia gibsoni)heat shock protein 70定序結果及NCBI blast結果的示意圖。 Figure 37 is a schematic diagram showing the results of sequencing of heat shock protein 70 of Babesia gibsoni and NCBI blast results.
<110> 基龍米克斯生物科技股份有限公司 <110> Kelong Meikes Biotechnology Co., Ltd.
<120> 區分犬焦蟲症分型的方法 <120> Method for distinguishing the classification of canine worms
<130> 101B0405 <130> 101B0405
<160> 4 <160> 4
<170> 派登印版本3.5(PatentIn version 3.5) <170> Pai Printing Version 3.5 (PatentIn version 3.5)
<210> 1 <210> 1
<211> 1944 <211> 1944
<212> DNA <212> DNA
<213> 犬焦蟲(Babesia canis) <213> Babesia canis
<220> <220>
<221> 基因 <221> gene
<222> (1)..(1944) <222> (1)..(1944)
<400> 1 <400> 1
<210> 2 <210> 2
<211> 1941 <211> 1941
<212> DNA <212> DNA
<213> 吉布松焦蟲(Babesia gibsoni) <213> Gibson gibsoni ( Babesia gibsoni )
<220> <220>
<221> 基因 <221> gene
<222> (1)..(1941) <222> (1)..(1941)
<400> 2 <400> 2
<210> 3 <210> 3
<211> 18 <211> 18
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 無 <223> None
<220> <220>
<221> 其他功能 <221> Other features
<222> (1)..(18) <222> (1)..(18)
<223> 引子BCBghsp231F <223> Introduction BCBghsp231F
<400> 3 <400> 3
<210> 4 <210> 4
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 無 <223> None
<220> <220>
<221> 其他功能 <221> Other features
<222> (1)..(20) <222> (1)..(20)
<223> 引子BCBghsp231R;r=g or a;y=t/u or c;k=g or t/u <223> Introduction BCBghsp231R;r=g or a;y=t/u or c;k=g or t/u
<400> 4 <400> 4
Claims (5)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW102100134A TWI532844B (en) | 2013-01-03 | 2013-01-03 | Method of distinguishing dog coccidia |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
TW102100134A TWI532844B (en) | 2013-01-03 | 2013-01-03 | Method of distinguishing dog coccidia |
Publications (2)
Publication Number | Publication Date |
---|---|
TW201428108A TW201428108A (en) | 2014-07-16 |
TWI532844B true TWI532844B (en) | 2016-05-11 |
Family
ID=51725982
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW102100134A TWI532844B (en) | 2013-01-03 | 2013-01-03 | Method of distinguishing dog coccidia |
Country Status (1)
Country | Link |
---|---|
TW (1) | TWI532844B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109136398A (en) * | 2018-09-15 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of detection dog Babesia kit and detection method |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG10201607084XA (en) * | 2016-08-25 | 2018-03-28 | Delta Electronics Intl Singapore Pte Ltd | Primer Pair, Kit And Method Of Detecting Babesia Canis |
CN108359722B (en) * | 2017-01-26 | 2022-04-08 | 艾迪克斯基因科技股份有限公司 | Integrated system and method for monitoring dynamic change of microorganism |
-
2013
- 2013-01-03 TW TW102100134A patent/TWI532844B/en active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109136398A (en) * | 2018-09-15 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of detection dog Babesia kit and detection method |
Also Published As
Publication number | Publication date |
---|---|
TW201428108A (en) | 2014-07-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Tanrıverdi et al. | Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis | |
Demeler et al. | Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR | |
Valasek et al. | The power of real-time PCR | |
Thekisoe et al. | Evaluation of loop-mediated isothermal amplification (LAMP), PCR and parasitological tests for detection of Trypanosoma evansi in experimentally infected pigs | |
Modak et al. | Rapid point-of-care isothermal amplification assay for the detection of malaria without nucleic acid purification | |
CN104046700B (en) | The detection kit of a kind of Rapid identification donkey hide, horse skin and mule skin | |
de Almeida et al. | Detection and differentiation of Leishmania spp. in clinical specimens by use of a SYBR green-based real-time PCR assay | |
US11773454B2 (en) | Multiplex PCR amplification method for species and human individual recognition and identification of unknown biological sample suspected to be from human | |
Rahumatullah et al. | Triplex PCR using new primers for the detection of Toxoplasma gondii | |
CN113957172B (en) | Kit for simultaneously detecting various cat pathogens and detection method and application thereof | |
da Silva et al. | Optimization of single-tube nested PCR for the diagnosis of visceral leishmaniasis | |
TWI532844B (en) | Method of distinguishing dog coccidia | |
Sunnotel et al. | Rapid and sensitive detection of single Cryptosporidium oocysts from archived glass slides | |
CN108486223B (en) | Babesia gibsoni RPA molecular detection method | |
Switaj et al. | Association of ocular toxoplasmosis with type I Toxoplasma gondii strains: direct genotyping from peripheral blood samples | |
US20080268425A1 (en) | Methods and kits for detecting classical swine fever virus | |
WO2021188935A1 (en) | Materials and methods for detecting coronavirus | |
Airs et al. | Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa | |
Meyer et al. | Detection of Koi herpesvirus: impact of extraction method, primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations | |
Dai et al. | Early detection of Toxoplasma gondii infection in Mongolian gerbil by quantitative real-time PCR | |
Fernandez‐Senac et al. | A comparison of the use of different swab materials for optimal diagnosis of amoebic gill disease (AGD) in Atlantic salmon (Salmo salar L.) | |
Ghasemian et al. | SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples | |
Xiang et al. | A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples | |
CN110592269A (en) | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) | |
CN110878380B (en) | Primer composition, kit and method for detecting vesicular stomatitis virus Indiana type and new Jersey type |