本文選題：腹瀉病原體 + HAND系統(tǒng)��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景和目的 急性感染性腹瀉(簡稱腹瀉)一直是危害人類健康特別是嬰幼兒健康的常見病和多發(fā)病,是全球范圍內(nèi)影響兒童和成人的重要公共衛(wèi)生問題。引起腹瀉的病原體主要是病毒和細(xì)菌,但具體種類繁多,且常常伴有多種腹瀉病原體復(fù)合感染的情況,在實(shí)際檢測中往往需要從這一系列的懷疑對象中確定腹瀉病原體,為腹瀉病的快速診斷帶來了極大的困擾。因此,建立一種高通量、高效的腹瀉病原體快速檢測技術(shù)對于診斷、控制和及時(shí)治療腹瀉病具有重要意義。然而,國內(nèi)外對腹瀉病原體實(shí)驗(yàn)室檢測還很大程度依賴傳統(tǒng)分離培養(yǎng)和生化鑒定,但是分離培養(yǎng)和生化鑒定敏感性低、費(fèi)時(shí)費(fèi)力,需要一周左右才能完成,難以達(dá)到暴發(fā)流行時(shí)快速檢測的要求,而且有的病原體(如諾如病毒)目前還沒有合適的細(xì)胞體系和動(dòng)物模型進(jìn)行培養(yǎng)。一些免疫學(xué)方法(如酶聯(lián)免疫吸附試驗(yàn))也被廣泛的應(yīng)用到腹瀉病原體的檢測,但其敏感性低,假陰性率高,容易造成漏檢,而且對于細(xì)菌來說需要預(yù)先將樣本中的目標(biāo)菌進(jìn)行濃縮和純化,短時(shí)間內(nèi)也很難得到結(jié)果,難以達(dá)到快速檢測的要求。隨著分子生物學(xué)檢測方法的發(fā)展,基于腹瀉病原體的分子生物學(xué)診斷技術(shù)如PCR、RT-PCR、Real-Time PCR、LAMP等在腹瀉病原體的檢測以及腹瀉病診斷方面發(fā)揮著重大作用,克服了以上的缺點(diǎn),特異性和敏感性較高,已成為目前腹瀉病原體快速診斷的常規(guī)手段,但這些方法一次只能檢測一種病原體,達(dá)不到高通量的檢測要求,對于復(fù)合感染的病例也容易漏檢,而且Real-Time PCR儀器費(fèi)用昂貴,檢測成本較高,操作復(fù)雜,不利于在基層實(shí)驗(yàn)室推廣。基因芯片技術(shù)雖然具有高通量的特點(diǎn),但也存在技術(shù)成本昂貴、復(fù)雜、檢測靈敏度低且重復(fù)性差等難以解決的問題。多重PCR是一種相對省時(shí)、省力方法,可以在一個(gè)反應(yīng)體系中同時(shí)檢測多個(gè)病原體,具有高通量、低成本的特點(diǎn),但由于多重PCR是多種不同引物混合,容易形成引物間的相互干擾和引物二聚體,使得反應(yīng)體系擴(kuò)增效率不均衡,穩(wěn)定性差,而基于HAND系統(tǒng)多重PCR可以有效的解決這些問題。 相同標(biāo)簽輔助-無引物二聚體(Homo-Tag Assisted Non-Dimer,HAND)系統(tǒng),亦稱同源加尾系統(tǒng),是1997年Browine為了消除PCR中引物二聚體的產(chǎn)生而設(shè)計(jì)的實(shí)驗(yàn)方法。該系統(tǒng)采用兩種引物,即加尾引物(3’端為特異性結(jié)合序列,5’端為添加的通用尾巴序列)和尾巴引物(序列與加尾引物5’端添加的通用尾巴相同)。其基本原理首先是低濃度的加尾引物與模板特異性結(jié)合,擴(kuò)增形成兩端均含有尾巴引物相同序列的初始PCR產(chǎn)物；此時(shí)若加尾引物之間形成引物二聚體,由于兩端存在互補(bǔ)的序列,引物二聚體的單鏈會(huì)各自形成一個(gè)穩(wěn)定的發(fā)夾結(jié)構(gòu),而該結(jié)構(gòu)難以成為下一循環(huán)的擴(kuò)增模板,從而大大的減少了引物二聚體的形成；其次是待低濃度的加尾引物消耗完,高濃度的尾巴引物以初始PCR產(chǎn)物為模板進(jìn)行特異性結(jié)合,直至擴(kuò)增完成。所以將HAND系統(tǒng)與多重PCR結(jié)合的優(yōu)點(diǎn)是可以有效地抑制引物二聚體的產(chǎn)生,提高多重PCR體系引物的容納數(shù)量,減小各基因擴(kuò)增效率的差異,使擴(kuò)增均衡高效和穩(wěn)定。因此,本研究第一部分?jǐn)M構(gòu)建針對常見腹瀉病毒、腸道致病菌、致病性弧菌三組腹瀉病原體的基于HAND系統(tǒng)多重PCR檢測方法,旨在為腹瀉病的診斷尋求廣譜高效的快速檢測方法。 弧菌是一類菌體短小,直或彎曲狀的革蘭氏陰性細(xì)菌,兼性厭氧,廣泛分布于自然界,以淡水及海水中最多�；【泻芏喾N,其中已經(jīng)證明對人類具有致病性的弧菌主要有霍亂弧菌(vibrio cholera)、副溶血弧菌(vibrioparahaemolyticus)、創(chuàng)傷弧菌(vibrio vulnificus)、擬態(tài)弧菌(vibrio mimicus)和溶藻弧菌(vibrio alginolyticus)等。致病性弧菌廣泛存在于溫帶和熱帶地區(qū)的海水、海底沉積物、浮游生物和水產(chǎn)品中,而因各種致病性弧菌污染水產(chǎn)品導(dǎo)致的問題也越來越多。人類可以通過食用被弧菌污染的水產(chǎn)品而造成急性胃腸炎和敗血癥等疾病,在影響人類的健康同時(shí)還可能因弧菌污染影響水產(chǎn)品養(yǎng)殖業(yè)的發(fā)展。此外,水產(chǎn)品中的分離的致病性弧菌大多數(shù)是非流行株,但它們卻是一些弧菌毒素基因的保存庫,而這些毒素基因的水平轉(zhuǎn)移可能造成非流行株與流行株的轉(zhuǎn)變。因此,開展致病性弧菌的分子流行病學(xué)調(diào)查,可為防控致病性弧菌感染引起的疾病提供有效信息�；诖�,本研究第二部分?jǐn)M利用構(gòu)建的基于HAND系統(tǒng)致病性弧菌多重PCR方法對2012.8—2013.7期間珠海、中山兩地供澳水產(chǎn)品中的致病性弧菌進(jìn)行描述性研究,并了解弧菌的相關(guān)毒素基因的分布。 方法 1.基于HAND系統(tǒng)腹瀉病原體多重PCR檢測方法的建立 首先選擇A組輪狀病毒的VP6基因、諾如病毒的RDRP基因、星狀病毒的NSP基因、札如病毒的caspid基因作為4種常見腹瀉病毒的靶基因,選擇志賀氏菌的virA基因、沙門氏菌的invA基因、小腸結(jié)腸炎耶爾森菌的ail基因、金黃色葡萄球菌的nuc基因、大腸桿菌0157：H7的rfbE基因作為5種腸道致病菌的靶基因,選擇創(chuàng)傷弧菌的vvhA基因、霍亂弧菌的ompW基因、副溶血弧菌的toxR基因、擬態(tài)弧菌的VMH基因、溶藻弧菌的gyrB基因作為5種致病性弧菌的靶基因,根據(jù)靶基因的保守序列設(shè)計(jì)特異性引物并在5’端加上尾巴序列,通過優(yōu)化加尾引物和尾巴引物的濃度、Mg2+濃度、DMSO濃度、循環(huán)參數(shù)構(gòu)建三組基于HAND系統(tǒng)腹瀉病原體多重PCR反應(yīng)體系,再分析其穩(wěn)定性、特異性和檢測下限值,并運(yùn)用于模擬的臨床糞便樣本檢測,盲法試驗(yàn)評價(jià)方法的實(shí)用性。 2.供澳水產(chǎn)品中致病性弧菌分子流行病學(xué)調(diào)查 2012.8--2013.7期間,采用隨機(jī)抽樣的方法,每月抽取大約125份珠海、中山兩地的供澳水產(chǎn)品樣,利用建立的基于HAND系統(tǒng)致病性弧菌多重PCR方法進(jìn)行初篩,再將初篩陽性樣品在TCBS培養(yǎng)上分離培養(yǎng)和生化鑒定進(jìn)行驗(yàn)證,所得結(jié)果進(jìn)行致病性弧菌的季節(jié)性、地域性和水產(chǎn)品種類分析；對分離到的陽性菌株利用PCR方法檢測ctxA、zot、ace、tcpA、tdh、trh?tlh、viuB8個(gè)弧菌毒素基因。 結(jié)果 1.基于HAND系統(tǒng)腹瀉病原體多重PCR檢測方法的建立 (1)成功構(gòu)建基于HAND系統(tǒng)腹瀉病毒多重RT-PCR檢測方法。特異性分析顯示四種腹瀉病毒間無交叉反應(yīng),敏感性分析顯示輪狀病毒、諾如病毒、星狀病毒和札如病毒的檢測下限分別達(dá)到48、9.6、1.92和48pg；盲法試驗(yàn)評價(jià)結(jié)果顯示符合率100%； (2)成功構(gòu)建基于HAND系統(tǒng)腸道致病菌多重PCR檢測方法。特異性分析顯示五種腸道致病菌無交叉反應(yīng),特異性100%,敏感性分析顯示志賀氏菌、大腸桿菌0157：H7、小腸結(jié)腸炎耶爾森菌三種致病菌的檢測下限值均為100cfu/ml,金黃色葡萄球菌的檢測下限值為1000cfu/ml,沙門氏菌的檢測下限值為10cfu/ml；盲法試驗(yàn)評價(jià)結(jié)果顯示符合率100%； (3)成功構(gòu)建基于HAND系統(tǒng)致病性弧菌多重PCR檢測方法。特異性分析顯示五種致病性弧菌無交叉反應(yīng),特異性100%,敏感性分析顯示創(chuàng)傷弧菌、霍亂弧菌、副溶血弧菌、溶藻弧菌四種致病性弧菌的檢測下限值均為100cfu/ml,擬態(tài)弧菌的檢測下限值為10cfu/ml；盲法試驗(yàn)評價(jià)結(jié)果顯示符合率100%。 2.供澳水產(chǎn)品中致病性弧菌分子流行病學(xué)特征 (1)基本情況：共收集1510份供澳水產(chǎn)品樣本,檢出898份陽性,弧菌陽性率為59.5%；復(fù)合感染樣本563份,復(fù)合感染率37.3%,其中副溶血弧菌和溶藻弧菌復(fù)合感染樣本293份,占52%;具體霍亂弧菌、副溶血弧菌、創(chuàng)傷弧菌、擬態(tài)弧菌和溶藻弧菌的陽性數(shù)分別為：329份(21.8%)、535份(35.4%)、56份(3.7%)、108份(7.2%)、611份(40.5%),其中霍亂弧菌中僅有2株O1群小川型,5株O1群稻葉型,其余為非O1/非0139群； (2)三間分布：在一年中的5-11月份致病性弧菌總陽性率較高,而1月份和12月份的致病性弧菌總陽性率較低,一年中每月弧菌的陽性率差異有統(tǒng)計(jì)學(xué)意義(χ2=198.26,P=0.000),供澳水產(chǎn)品中致病性弧菌主要在夏秋季流行；珠海地區(qū)總弧菌陽性率69.5%高于中山的51.9%,兩地總弧菌陽性率差異有統(tǒng)計(jì)學(xué)意義(χ2=47.42,P=0.000),珠海供澳水產(chǎn)品中弧菌的污染現(xiàn)象比中山地區(qū)嚴(yán)重；貝類的總弧菌陽性率70.6%高于魚類的54.4%,兩類水產(chǎn)品總弧菌陽性率差異有統(tǒng)計(jì)學(xué)意義(χ2=36.65,P=0.000),貝類中致病性弧菌的污染現(xiàn)象比魚類嚴(yán)重； (3)毒素基因分布：329株霍亂弧菌中,ctxA均為陰性,1株ace+、zot+、tcpA+14株ace+、zot+、tcpA-,6株ace-、zot+、tcpA-,5株ace-、zot-、tcpA+；535株副溶血弧菌中,tlh均陽性,7株tdh陽性,檢出率1.3%；9株trh陽性,檢出率1.6%；56株創(chuàng)傷弧菌中,12株viuB陽性,檢出率21.4%；108株擬態(tài)弧菌中,ctxA、ace、tcpA均陰性,2株zot陽性；611株溶藻弧菌中,146株tlh陽性,檢出率23.9%,tdh、trh均為陰性。 結(jié)論 1.所建立的三組基于HAND系統(tǒng)腹瀉病原體多重PCR檢測方法,具有快速、穩(wěn)定、特異、靈敏和低成本的特點(diǎn),非常適用于基層醫(yī)學(xué)實(shí)驗(yàn)室； 2.供澳水產(chǎn)品中致病性弧菌污染的現(xiàn)象比較嚴(yán)重,且普遍存在兩種或三種致病性弧菌的多重污染,要加大致病性弧菌的監(jiān)測力度,以防致病性弧菌的感染和流行； 3.環(huán)境中分離的致病性弧菌大多數(shù)是非流行株的,卻是已知毒素基因的重要保存庫。
[Abstract]:Background and purpose of research
Acute infectious diarrhea (abbreviated diarrhoea) is a common and frequently occurring disease that endangers human health, especially infant health. It is an important public health problem that affects children and adults worldwide. The pathogens causing diarrhoea are mainly viruses and bacteria, but the specific species are complex and often accompanied by multiple diarrhea pathogens compound infection. In actual testing, it is often necessary to identify diarrhoea pathogens from this series of sceptics and cause great trouble for the rapid diagnosis of diarrhoea. Therefore, the establishment of a high throughput and efficient detection technique for diarrhea pathogens is of great significance for the diagnosis, control and treatment of diarrhoea. Laboratory tests for diarrhoea pathogens are also largely dependent on traditional isolation and biochemical identification, but the isolation, culture and biochemical identification are low and time-consuming. It takes about a week to complete, and it is difficult to meet the requirements of rapid detection when the outbreak is outburst, and some pathogens (such as norovirus) have no proper cell system at present. Some immunological methods, such as enzyme linked immunosorbent assay (ELISA), are also widely used in the detection of diarrhoea pathogens, but their sensitivity is low, the false negative rate is high, and it is easy to cause leakage, and for bacteria, it is necessary to concentrate and purify the target bacteria in advance, and it is difficult to get the result in a short time. With the development of molecular biological detection methods, molecular biological diagnostic techniques based on diarrhoea, such as PCR, RT-PCR, Real-Time PCR and LAMP, have played a major role in the detection of diarrhoea pathogens and the diagnosis of diarrhoea, which have overcome the above shortcomings, and have become more specific and sensitive. It is a routine method for the rapid diagnosis of diarrhoea pathogens, but these methods can only detect one kind of pathogen at a time, can not meet the requirements of high flux detection, and it is easy to leak detection for the cases of complex infection, and the Real-Time PCR instrument is expensive, the cost is high, the operation is complex, and it is not conducive to the promotion of the basic laboratory. Although the operation is characterized by high throughput, there are also difficult problems such as high cost, complexity, low sensitivity and poor repeatability. Multiple PCR is a relatively time-saving and labor-saving method to detect multiple pathogens at the same time in a reaction system, with high flux and low cost, but multiple PCR is different With the same primers, the interaction between primers and primer two polymer can be easily formed, which makes the efficiency of the reaction system uneven and the stability is poor, and the multiple PCR based on HAND system can effectively solve these problems.
The same label auxiliary Homo-Tag Assisted Non-Dimer (HAND) system, also known as the homologous tailing system, is an experimental method designed by Browine in 1997 to eliminate the production of primer two polymer in PCR. The system uses two primers, namely, the tail primer (3 'end as a specific binding sequence, and the 5 "end as a general tail order added. Column) and tail primers (the same sequence is the same as the general tail added to the 5 'end of the tail primer). The basic principle is that a low concentration of tail primers and a template specific binding are first amplified to form an initial PCR product at both ends containing the same sequence of the tail primers; at this time, a primer two polymer is formed between the tailed primers, because the two ends are complementary to each other. Sequence, the single strand of primer two polymer will each form a stable hairpin structure, and the structure is difficult to be an amplification template for the next cycle, thus greatly reducing the formation of primer two polymer; secondly, the tail primer for low concentration is consumed, and the high concentration tail primers specifically combine with the initial PCR product as a template. The combination of HAND system and multiple PCR can effectively inhibit the production of primer two polymer, increase the number of primers in multiple PCR system, reduce the difference of gene amplification efficiency and make the amplification balanced and efficient and stable. Therefore, the first part of this study is to construct the common diarrhea virus and intestinal pathogenic bacteria. The multiplex PCR detection method based on HAND system for diarrheal pathogens in three groups of pathogenic vibrio is aimed at finding a broad spectrum and efficient method for the diagnosis of diarrheal diseases.
Vibrio is a group of Gram-negative bacteria with short, straight or curved form of gram negative bacteria. It is widely distributed in nature and is widely distributed in the natural world. There are many Vibrio species in fresh water and sea water. Among them, Vibrio has been proved to be pathogenic Vibrio (Vibrio cholera), Vibrio parahaemolyticus (vibrioparahaemolyticus), Vibrio vulnificus (vibr). IO vulnificus), Vibrio mimetic (Vibrio mimicus) and Vibrio alginolyticus (Vibrio alginolyticus). Pathogenic vibrio is widely found in temperate and tropical seawater, seafloor sediments, plankton and aquatic products, and more and more problems are caused by various pathogenic Vibrio contaminated aquatic products. Human can be contaminated by Vibrio. Diseases such as acute gastroenteritis and septicemia caused by dyed aquatic products may affect human health and may also affect the development of aquatic products in aquatic products. In addition, the isolated Vibrio isolates in aquatic products are mostly non epidemic strains, but they are the storage of some Vibrio toxin genes, and the water of these toxin genes. Therefore, the molecular epidemiological investigation of pathogenic Vibrio can provide effective information for preventing and controlling the disease caused by Vibrio Vibrio infection. Based on this, the second part of this study is to use the constructed multiple PCR method of pathogenic Vibrio based on HAND system for the period of 2012.8 to 2013.7. A descriptive study of pathogenic Vibrio was carried out in Zhuhai and Zhongshan, and the distribution of Vibrio related toxin genes was also studied.
Method
1. establishment of multiplex PCR assay for diarrhea pathogens based on HAND system
First, select the VP6 gene of A rotavirus, the RDRP gene of norovirus, the NSP gene of the stellate virus, the caspid gene of the zzavirus as the target gene for 4 common diarrhea viruses, select the virA gene of Shigella, the invA gene of Salmonella, the ail gene of Jerson bacteria of the enterocolitis, the NUC gene of Staphylococcus aureus, and the large intestine The rfbE gene of bacilli 0157:H7 was used as the target gene for 5 kinds of intestinal pathogenic bacteria, which chose the vvhA gene of Vibrio vulnificus, the ompW gene of Vibrio cholerae, the toxR gene of Vibrio parahaemolyticus, the VMH gene of Vibrio mimici, the gyrB gene of Vibrio alginolyticus as the target gene of 5 pathogenic Vibrio, and the specific primers were designed according to the conservative sequence of the target gene and 5 By adding tail sequence, three groups of multiple PCR reaction systems based on HAND system diarrhea pathogen were constructed by optimizing the concentration of tail and tail primers, concentration of Mg2+, DMSO concentration, and cycle parameters, and then the stability, specificity and detection limit were analyzed, and used for simulated clinical fecal sample detection, and the method of blind test evaluation was real. Use sex.
2. molecular epidemiology of pathogenic Vibrio in Australian aquatic products
During the period of 2012.8--2013.7, a random sampling method was used to extract about 125 seafood samples from Zhuhai and Zhongshan each month. Using the established multiple PCR method based on HAND system pathogenic Vibrio, the initial sifting samples were screened in TCBS culture and verified by biochemical identification. The results were pathogeny arc. The seasonal, regional and aquatic products of the bacteria were analyzed, and the isolates were detected by PCR method to detect ctxA, zot, ACE, tcpA, TDH, TRH? TLH, and viuB8 Vibrio toxin genes.
Result
1. establishment of multiplex PCR assay for diarrhea pathogens based on HAND system
(1) successful construction of multiple RT-PCR detection methods based on HAND system diarrhea virus. Specific analysis showed no cross reaction between four kinds of diarrhea viruses. Sensitivity analysis showed that rotavirus, norovirus, stellate virus and zzavirus had a lower limit of 48,9.6,1.92 and 48pg, and the result of blind test showed that the coincidence rate was 100%.
(2) successful construction of multiple PCR detection methods for intestinal pathogenic bacteria based on HAND system. Specific analysis showed that five kinds of intestinal pathogenic bacteria had no cross reaction, specificity 100%. Sensitivity analysis showed that Shigella, Escherichia coli 0157:H7, and three pathogenic bacteria of Jerson bacteria of enterocolitis were both 100cfu/ml and Staphylococcus aureus. The lower limit is 1000cfu/ml, the lower limit of Salmonella detection is 10cfu/ml, and the result of blind test shows that the coincidence rate is 100%.
(3) successful construction of multiple PCR detection methods based on HAND pathogenic Vibrio. Specific analysis showed that five pathogenic Vibrio without cross reaction, specificity 100%, sensitivity analysis showed that the detection limit of Vibrio vulnificus, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio alginolyticus were 100cfu/ml, and the detection limit of Vibrio mimicus The value is 10cfu/ml; the blind test results show that the coincidence rate is 100%..
2. molecular epidemiology of pathogenic Vibrio in Australian aquatic products
(1) the basic situation: a total of 1510 samples of Australian aquatic products were collected, 898 positive were detected, the positive rate of Vibrio was 59.5%, 563 samples of compound infection and 37.3% compound infection rate, including 293 samples of Vibrio parahaemolyticus and Vibrio alginolyticus, 52%, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio Vibrio and Vibrio alginolyticus The numbers were 329 (21.8%), 535 (35.4%), 56 (3.7%), 108 (7.2%) and 611 (40.5%), of which only 2 O1 group of Vibrio cholerae, 5 O1 group rice leaf type, and the other non O1/ non 0139 group;
(2) three distribution: the total positive rate of Vibrio virulence was higher in the month of 5-11, and the total positive rate of pathogenic Vibrio in January and December was lower, and the positive rate of Vibrio was statistically significant in one year (x 2=198.26, P=0.000). The pathogenic Vibrio in Australian aquatic products was mainly in summer and autumn, and the total Vibrio Yang in Zhuhai area The rate of sex was 69.5% higher than that of 51.9% in Zhongshan. The positive rate of Vibrio was statistically significant (x 2=47.42, P=0.000). The pollution of Vibrio in Zhuhai aquatic products was more serious than that in Zhongshan; the positive rate of the total Vibrio in shellfish was 70.6% higher than that of fish, and the positive rate of total arc bacteria in the two types of aquatic products was statistically significant (x 2=36.65, P=0.000). The contamination of pathogenic Vibrio in shellfish is more serious than that in fish.
(3) distribution of toxin gene: among 329 strains of Vibrio cholerae, ctxA was negative, 1 strains of ace+, zot+, tcpA+14 strain ace+, zot+, tcpA-, 6 ace-, zot+, tcpA-, 5 strains of ace-, 535 strains of Vibrio parahaemolyticus, 7 positive, detection rate 1.3%, 9 strains positive, 1.6%, 12, 12, 21.4%; 21.4%; 21.4%; 12 Among the 8 Vibrio mimicus, ctxA, ACE, tcpA were all negative, 2 zot positive, 611 strains of Vibrio alginolyticus, 146 TLH positive, the detection rate was 23.9%, TDH and TRH were negative.
conclusion
1. methods of multiple PCR detection based on HAND system for diarrhea pathogens were established, which have the characteristics of fast, stable, specific, sensitive and low cost, and are very suitable for the basic medical laboratory.
2. the contamination of pathogenic Vibrio in Australian aquatic products is serious, and the multiple contamination of two or three pathogenic vibrio is widespread, and the monitoring of Vibrio vulnificus should be added in order to prevent the infection and epidemic of Vibrio.
3. most pathogenic Vibrio isolated from the environment are non epidemic strains, but are important repositories for known toxin genes.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.5

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