聚合酶链式反应热流变化的DSC实验研究

在PCR每个循环中,目的基因在DNA聚合酶的催化作用下实现快速扩增,同时伴随着化学键的断裂和生成,而不同循环数的扩增效率不同,引起的热现象也不同。实验通过差示扫描量技术,以HBV为PCR扩增体系,分别研究了变性、退火和延伸阶段的热焓及其随循环数的变化,通过分析得出：变性阶段是放热过程,第17个循环放热量达到最大,退火和延伸阶段是吸热过程;3个阶段的热焓随循环数增加都发生明显的变化,其中变性阶段的热流变化最关键。     

胚胎发育早期转基因整合的研究

转基因动物的建立是一项复杂而艰苦的工作,在转基因胚移植受体前对其进行检测,无疑对转基因动物建立具有重要意义。使用小鼠乳清酸蛋白（WAP）控制下的人粒细胞激落刺激因子（G-CSF）基因为显微注射片段,采用PCR方法检测了转基因胚,在1、2和8细胞期的阳性率为100％、77．7％和44．4％。为消除PCR扩增中的假阳性结果,构建了两个具有部分同源性的亚克隆片段进行共注射。PCR扩增片段跨越这一同源区域,转基因的非整合胚不能扩增出特异性片段。结果表明,1、2和8细胞期的阳性率分别为11．1％、55．5％和44．4％,较常规PCR检测获得更为明确的结论,为在大动物转基因的检测提供了新依据。     

瓯江2种(鱼骨)的ISSR分析优化和自然种群遗传多样性的研究

以唇[鱼骨]和花[鱼骨]为材料,对影响PCR反应的主要因子-模板DNA、引物、dNTPs、Mg2＋、Taq酶浓度进行优化,建立了适合唇[鱼骨]和花[鱼骨]基因组DNA的ISSR—PCR反应体系。25灿L的PCR反应液含有的组分和终浓度分别为：约60ng模板DNA、1．2UTaq酶、0．4μmol／L引物、2．0mmol／LMgCl,、0．2mmol／LdNTPs、2％甲酰胺。利用优化反应体系从60个ISSR引物中,筛选出18个稳定性高、重复性好的引物,对25个唇[鱼骨]个体和22个花[鱼骨]个体的DNA进行正式扩增,分别从两个种群分别扩增到168和156条扩增谱带,扩增片段长度在200--1500bp之间。根据扩增结果计算出两个种群的多态位点比例分别为67．52％和53．21％,Shannon多样性指数分别为0．3450和0．2749,Nei’s指数分别为0．2292和0．1837,平均有效等位基因数分别为1．3875和1．3169。两个种群内样本间的平均遗传距离分别为0．2129和0．1529,结果表明两个种群的遗传多态性较高,其中唇[鱼骨]的多样性明显高于花[鱼骨]群体。引物UBC845扩增的610bp带和UBC854扩增的1240bp带为唇[鱼骨]种的特异性谱带。[鱼骨]属鱼类基因组中均含有微卫星DNA序列：（AG）8、（GA）8、（CT）8和（GAA）8。本研究为唇[鱼骨]和花[鱼骨]的种质保护、合理开发利用以及选择育种提供了一些基础数据。     

香蕉RAPD分析初步研究

比较了不同提取方法对香蕉植株不同部位组织提取 DNA的质量及其 PCR扩增结果 ,对香蕉 RAPD分析中引物种类和浓度 ,复性温度 ,d NTPs,Taq DNA聚合酶浓度 ,热循环数等因素进行了比较影响分析。结果表明 ,虽然改良的 SDS法、CTAB法和 PVP法提取的植株嫩叶和吸芽 DNA提取量和纯度各不相同 ,但其 PCR扩增结果基本相同 ;相同克隆不同植株的 DNA其 PCR扩增结果也基本相同 ;建立了适合香蕉大规模 DNA多态性分析 RAPD反应体系 :2 5 μL反应液中 ,含 1倍缓冲液 ,0 .2 m Md NTPs,0 .3 2 p M随机引物 ,IUTaq酶 ,2 0 ng模板 DNA;反应循环数为 45 ,热循环条件为 94°C,1 min;3 7°C,1 min;72°C,1 .5 min;之前为 94°C,5 min;之后为72°C,1 0 min。在筛选的 2 49个随机引物中 ,有 1 8个在 7个品种上都能扩增出 3～ 1 0条比较清晰条带。     

TaqMan荧光定量RT-PCR检测水稻RAcl基因mRNA方法的建立

构建包含RAcl基因cDNA片段的质粒,作为水稻肌动蛋白基因RAcl之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参。从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAcl基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。建立了RAcl基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102—1护拷贝,阈值循环数（Ct）与PCR体系中起始模板量的对数值之间有着良好的线性关系（r=1．000）,扩增效率高（E=98．2％）。建立了基因RAcl实时定量PCR的质粒标准品。     

肿瘤干细胞标记物CD133与miR-429在大肠癌组织中的表达及其相关性研究

目的：检测CD133不同亚群大肠癌细胞HT-29的miR-429表达情况,探讨miR-429及CD133的表达与肿瘤的发生发展之间的关系。方法：采用荧光活化细胞分选法（FACS）分选出CD133不同亚群细胞,实时荧光定量PCR分别检测两组细胞miR-429的表达,合成miR-429寡核苷酸和阴性对照miRNA并分别转染CD133＋和CD133＋两个亚群细胞。再将细胞种植于非肥胖糖尿病／严重联合免疫缺陷（NOD／SCID）小鼠体内构建移植瘤模型,不同时间测量肿瘤体积和重量,RT—PCR及蛋白质印迹检测CD133＋和CD133＋两组肿瘤CD133mRNA和蛋白质表达。结果：血清检出CD133＋细胞为67．9％,miR-429的表达量是CD133＋细胞的（1．83±0．91）倍（P〈0．05）,CD133＋比例与miR-429表达呈负相关（r=0．591,P〈0．05）;miR-429＋／CD133＋组的移植瘤体积及重量与对照组比较有统计学差异（P〈0．05）,且miR-429＋/CD133＋组成瘤时间较对照组晚约2周,但miR-429＋/CD133＋组的移植瘤CD133表达量低,与阴性对照组比较无明显差异（P〉0．05）。结论：miR-429可能作为CD133的负性调控因子,具有抑制肿瘤生长的作用,但miR-429与CD133在肿瘤发生、发展过程中的作用机制有待进一步研究阐明。     

喂养不耐受早产儿大便大肠埃希菌、粪肠球菌和肺炎克雷伯菌的数量变化

目的观察早产儿喂养不耐受肠道中大肠埃希菌、粪肠球菌和肺炎克雷伯菌的变化。方法采用实时荧光定量PCR技术,分别对15例喂养不耐受早产儿和15例喂养耐受早产儿（对照组）生后第一天,出现喂养不耐受,喂养不耐受恢复后大便标本中的大肠埃希菌、肺炎克雷伯菌和粪肠球菌进行定量分析。结果喂养不耐受组中大肠埃希菌的拷贝数对数值（lg copies／g）分别为2．62±0．22、5．47±1．28、3．04±0．70,对照组分别为2．56±0．19、2．82±0．4、2．80±0．39;肺炎克雷伯菌的拷贝数对数值（1gcopies／g）分别为4．37±0．22、6．56±O．27、4．17±0．27,对照组分别为4,35±0．22、4．19±0．14、4．15±0．25;粪肠球菌的拷贝数（copies／g）分别为79．17±93．46、42。84±47．57、101。68±43．78,对照组分别为70．16±78．41、740．05±657．71、104．57±38．39。出现喂养不耐受时,两组的3种细菌拷贝数比较差异具有统计学意义（P〈0．05）,而出现之前和恢复后两组细菌拷贝数差异无统计学意义（P〉0．05）。结论喂养不耐受时大肠埃希菌、肺炎克雷伯菌数量显著增高,可能参与喂养不耐受的发生,而粪肠球菌降低,可能起保护作用。     

头颈部放疗患者口腔假丝酵母菌感染的快速检测

目的通过对传统培养法和PCR法在假丝酵母菌感染检出率的比较,拟探索一种能够早期、快速、高效检测头颈部放疗患者假丝酵母菌感染的方法。方法收集120名头颈部放疗患者唾液,分别应用假丝酵母菌显色培养基（CHROMagar）进行分离、培养和鉴定;同时提取基因组DNA,通过假丝酵母菌通用引物、特异性引物、改良引物进行PCR扩增,结果与假丝酵母菌表型进行对比。结果与传统培养法相比,PCR法检出率更高（χ2=47.672,P=0.000）;改良特异性引物D扩增的检出率为77%,高于通用引物B（χ2=7.702,P=0.006）和特异性引物C（χ2=12.522,P=0.001）。结论本研究证实PCR技术耗时短,阳性检出率高,可用于头颈部肿瘤放疗患者假丝酵母菌感染的快速检测。     

FSH通过SATB1调控上皮性卵巢癌ES-2细胞的增殖和侵袭活性

目的：探讨特异AT序列结合蛋白1（special AT-rich sequence-bindingprotein,SATB1）在卵泡刺激素（Follicle stimulating hor-mone,FSH）诱导的上皮性卵巢癌ES-2细胞增殖和侵袭中的作用。方法：以Real-timePCR检测不同浓度FSH（0、10、20、40、80mlU／ml）处理后sATB1基因mRNA表达水平的变化。实验分4组：①siCon组,转染si-阴性对照（si-Negativecontrol）序列的实验组,对SATBl无干扰作用;②siSATB1组：转染特异性干扰下调SATB1的siSATB1序列;（3）FSH＋siCon组：以FSH处理的siCon组;（4）FSH＋siSATBl组：以FSH处理的sisATB1组。MTT法检测4组细胞的增殖情况,Westernblotting技术检测4组细胞细胞周期蛋白（CyclinDl）,基质金属蛋白酶2（MMP．2）的蛋白表达情况,Transwell侵袭实验检测4组细胞侵袭能力的变化。结果：1．FSH＋siCon组的细胞增殖能力明显高于siCon组的细胞增殖能力,FSH＋siCon组的CyclinD1蛋白相对表达量0．90＋0．08明显高于siCon组的0．37＋0．01（P均〈0．01）,提示FSH具有促进ES．2细胞增殖的作用。2．FSH＋siCon组的穿膜细胞数（30212）个明显高于siCon组（13919）个,FSH＋siCon组的MMP．2蛋白相对表达量0．40＋0．01明显高于siCon组的0．28＋0．02,提示FSH具有促进ES．2细胞侵袭能力的作用。3．随着FsH浓度的增高,SATBlmRNA的表达量逐渐增加,分别为1,1．66±0．04,1．79±0．21,2.31±0．03,以FsH浓度为80mlU／m1时最显著（P〈0．05）。4．FSH＋siSATBl组的细胞增殖能力明显低于FSH＋siCon组的细胞增殖能力,FSH＋siSATBl组的CyclinD1蛋白相对表达量0．22±0．02明显低于FSH＋siCon组的0．90±0．08（P均〈0．01）;FSH＋siSATB1组的穿膜细胞数（5216）个低于FSH＋siCon组的（30212）个,FSH＋siSATB1组的MMP-2蛋白相对表达量0．15±0．00明显低于FSH＋siCon组的0．40±0．01（P均〈0．01）,FSH促进ES-2细胞增殖和侵袭的能力由于SATB1基因表达的下降而被阻断。结论：SATBl是FSH作用的重要靶分子,介导FSH对上皮性卵巢癌ES-2细胞系增殖、侵袭活性的调控。     

荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列,经TA克隆构建重组质粒作为定量标准品,经FQ—PCR反应条件的优化及方法学评价,再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min;95℃ 20 s,60℃ 60 s（40 cycles）,20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％,批间CV值为1．96％;特异性较好;线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法,完全适用于临床检测。     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst . Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst . In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants.     

A cautionary note on the use of nested PCR for parasite screening--an example from avian blood parasites

The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these "false" Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these "false" Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.     

Development of a synthetic positive control which also detects plasmid contamination in diagnostic polymerase chain reaction

This paper describes a technique for the development of a positive control for use in a nested PCR to show that the PCR has worked correctly with both outer and inner primers designed for diagnostic amplification of 618 bp and 317 bp products respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: 1) making it visually easy to detect plasmid contamination and thus, prevent false positives from the plasmid; 2) develop a positive control when the target organism is at a very low prevalence so initial detection is not relied on for cloning positive controls. This will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives; or 3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrates how to use large oligonucleotide primers in PCR without non-specific binding occurring.     

Development of a Synthetic Positive Control Which Also Detects Plasmid Contamination in Diagnostic Polymerase Chain Reaction

This paper describes a technique for developing a positive control for use in a nested PCR to show that the PCR has functioned correctly with both outer and inner primers designed for the diagnostic amplification of 618 and 317 bp products, respectively. This positive control produces a larger product than the diagnostic sample that can be discriminated on an agarose gel. This technique is advantageous over traditional cloning of the diagnostic PCR product itself by: (1) making it visually easy to detect plasmid contamination and thus prevent false positives from the plasmid; (2) develop a positive control when the target organism is at a very low prevalence, so initial detection is not relied on for cloning positive controls (this will ensure the PCR is working correctly prior to diagnostic sampling, reducing false negatives); or (3) for developing a PCR and determining the sensitivity prior to the use of diagnostic samples. The methods used to produce this nested positive control demonstrate how to use large oligonucleotide primers in PCR without nonspecific binding occurring.     

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum .
Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum , which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.
Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.
Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.     

Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V.parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10⁷ CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.