Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

基于实时聚合酶链式反应检测特种乳中牛乳的掺伪

分别基于SYBR Green实时聚合酶链式反应（real-time polymerase chain reaction;real-time PCR）和TaqMan real-time PCR技术检测特种乳及其热处理加工产品中掺假的牛乳成分;同时探讨不同热处理方式对掺假检测的影响;以满足不同商品化特种乳制品的检测要求。结果表明;所设计的牛特异性引物可以扩增牛乳中的DNA;与非目标动物无交叉反应性;具有较高的特异性。两种real-time PCR方法对于牛乳DNA的检测限分别为1 pg（SYBR Green）和10 pg（TaqMan）;且均可以最低检测出特种乳混合物中0.1%（m/m）牛乳成分掺伪。为评估掺假模拟的重现性;在3 d内共进行了9 次重复实验;验证了方法具有较高的重复性。同时;为检测本研究中所建立实验方法的可用性;对20 种商业加工特种乳制品进行了真实性分析;其中在7 种产品中检测出牛乳成分;显示出所建立的检测方法具有较高的分辨率和实际应用价值。     

原料乳中嗜冷菌产脂肪酶条件的优化

研究了原料乳中嗜冷菌产生脂肪酶的几组影响条件。主要从pH值、培养温度、产酶培养基组成几个方面考虑。优化产酶条件，从而可提高脂肪酶的检测活力，为原料乳中嗜冷菌分泌的脂肪酶的研究奠定理论基础。     

水产品中创伤弧菌ddPCR定量方法的建立

选取创伤弧菌单拷贝基因met为靶基因,设计引物探针,建立对创伤弧菌准确定量的微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction,ddPCR）方法,并进行特异性、灵敏度和重复性实验,同时与实时PCR（real-time PCR）方法进行比较。结果显示,所建立的ddPCR方法可以快速、高效地检测出创伤弧菌,细菌纯培养物中其定量限可达323 拷贝数/mL,检测限可达61 拷贝数/mL,人工污染牡蛎样品中能最低能检测到1.13×102 拷贝数/g的目标菌。对人工污染样品中目标菌的检测,ddPCR的定量结果约为平板计数结果的1.4 倍,比real-time PCR方法的检测更加稳定准确。本研究建立的ddPCR检测方法能快速准确、特异、灵敏地定量检测创伤弧菌。     

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time <2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.     

多重real-time PCR技术快速鉴别特种乳中的乳源动物成分

建立一种特种乳中乳源动物成分快速鉴别多重实时聚合酶链式反应技术.通过筛选建立8种不同乳源物种检测的多重实时聚合酶链式反应方法,在一个反应体系里可同时检测4种乳源的特异性靶基因,绝对灵敏度达0.1～5 pg/μL,检出限可达0.1％.同时,建立了高效的DNA提取方法,样品裂解后可在12 min左右完成96个样品的DNA提...     

The effect of storage conditions on the composition and functional properties of blended bulk tank milk

The objective of this study was to investigate the effects of storage temperature and duration on the composition and functional properties of bulk tank milk when fresh milk was added to the bulk tank twice daily. The bulk tank milk temperature was set at each of 3 temperatures (2, 4, and 6°C) in each of 3 tanks on 2 occasions during two 6-wk periods. Period 1 was undertaken in August and September when all cows were in mid lactation, and period 2 was undertaken in October and November when all cows were in late lactation. Bulk tank milk stored at the 3 temperatures was sampled at 24-h intervals during storage periods of 0 to 96 h. Compositional parameters were measured for all bulk tank milk samples, including gross composition and quantification of nitrogen compounds, casein fractions, free amino acids, and Ca and P contents. The somatic cell count, heat stability, titratable acidity, and rennetability of bulk tank milk samples were also assessed. Almost all parameters differed between mid and late lactation; however, the interaction between lactation, storage temperature, and storage duration was significant for only 3 parameters: protein content and concentrations of free cysteic acid and free glutamic acid. The interaction between storage temperature and storage time was not significant for any parameter measured, and temperature had no effect on any parameter except lysine: lysine content was higher at 6°C than at 2°C. During 96 h of storage, the concentrations of some free amino acids (glutamic acid, lysine, and arginine) increased, which may indicate proteolytic activity during storage. Between 0 and 96 h, minimal deterioration was observed in functional properties (rennet coagulation time, curd firmness, and heat stability), which was most likely due to the dissociation of β-casein from the casein micelle, which can be reversed upon pasteurization. Thus, this study suggests that blended milk can be stored for up to 96 h at temperatures between 2°C and 6°C with little effect on its composition or functional properties.     

RT-FQPCR法快速检测葡萄酒中醋酸菌群

建立一种快速、灵敏的检测葡萄酒中醋酸菌群的检测技术。通过改进葡萄酒中脱氧核糖核酸（DNA）提取方法,设计醋酸菌通用引物,优化实时荧光定量聚合酶链式反应（RT-FQPCR）条件,确定葡萄酒中醋酸菌的快速检测方法,同时利用7种醋酸菌对该方法的通用性、特异性以及灵敏度进行了验证。结果表明,7种醋酸菌均具有明显扩增曲线;非目的菌在该种条件下未见扩增,综合检测灵敏度为124 CFU/mL。以10种市售葡萄酒为基质,在其中添加醋酸菌均可检测出目标添加菌株。该方法通用性强,可用于葡萄酒中醋酸菌群的检测。     

利用mPCR方法检测巴氏奶中致病菌的鲁棒性研究

为解决巴氏奶货架期短与致病菌传统检测方法耗时长相矛盾问题,建立一种检测巴氏奶中的阪崎克罗诺杆菌、大肠杆菌和沙门氏菌的鲁棒性多重聚合酶链式反应（multiple polymerase chain reaction,mPCR）方法。旨在常规mPCR的基础上深入研究,提高方法的准确性和稳定性。首先,针对每个目标菌株选择两种基因,并设计两组特异性引物,建立两套mPCR扩增体系,进行双重检测;然后,在不改变方法灵敏度的前提下,对优化了的退火温度进行范围稳定性选择;最后,将提出方法与国标方法GB 4789.40-2016、GB 4789.38-2012、GB 4789.4-2016进行比较,同时检测人工污染样品并评价应用效果。结果表明,第一套mPCR检测巴氏奶中三种致病菌可在退火温度59~59.5 ℃（温差0.5 ℃）的范围内,具有较好的检测准确性和稳定性,灵敏度为10 CFU/mL。第二套mPCR检测巴氏奶中三种致病菌可在退火温度57.5~58.5 ℃（温差1 ℃）的范围内,不影响方法的准确性和稳定性,灵敏度为10 CFU/mL。采用建立的鲁棒性mPCR方法对人工污染的50份巴氏奶样品进行检测,检测结果与国标方法一致。在检测时效上需要4 h,较国标方法（检测时间为62~148 h）显著（P<0.05）缩短检测时间。该方法对微生物流行病学调查和研究,尤其是对巴氏奶中致病菌的快速检测和安全控制具有一定的实际应用价值。     

Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China

Lipases secreted by psychrotrophic bacteria are known to be heat resistant and can remain active even after the thermal processing of milk products. Such enzymes are able to destabilize the quality of milk products by causing a rancid flavor. Rapid detection of a small amount of heat-resistant lipase-producing psychrotrophic bacteria is crucial for reducing their adverse effects on milk quality. In this study, we established and optimized a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Pseudomonas fluorescens in raw cow milk, as the most frequently reported heat-resistant lipase-producing bacterial species. Pseudomonas fluorescens-specific DNA primers for LAMP were designed based on the lipase gene sequence. Reaction conditions of the LAMP assay were tested and optimized. The detection limit of the optimized LAMP assay was found to be lower than that of a conventional PCR-based method. In pure culture, the detection limit of the LAMP assay was found to be 4.8 × 10¹ cfu/reaction of the template DNA, whereas the detection limit of the PCR method was 4.8 × 10² cfu/reaction. Evaluation of the performance of the method in P. fluorescens-contaminated pasteurized cow milk revealed a detection limit of 7.4 × 10¹ cfu/reaction, which was 10² lower than that of the PCR-based method. If further developed, the LAMP assay could offer a favorable on-farm alternative to existing technologies for the detection of psychotrophic bacterial contamination of milk, enabling improved quality control of milk and milk products.     

基于单拷贝核基因的荧光定量PCR技术检测驴奶的含量

本研究建立了一种基于内参基因的标准化实时聚合酶链式反应(real-time PCR)方法,能够定量检测混合掺假的灭菌乳中驴奶的含量。使用单拷贝核基因代替多拷贝线粒体基因,基于Ct值(驴特异性引物/内参引物)与驴奶含量的线性关系,对含量为5%~100% 的驴奶建立了标准曲线。该方法具有良好的线性相关(R²=0.9650)和较高的准确度,对含有20%、50%和80% 驴奶的模拟掺假样品进行定量分析,平均回收率为109.16%,平均CV值为4.68%。因此,该方法可以快速、准确地对驴奶进行定量,从而确定驴奶是否掺假以及掺假比例。

     

应用荧光聚合酶链式反应检测冷冻羊肉卷中羊肉的含量

目的：探索应用荧光聚合酶链式反应（polymerase chain reaction,PCR）法检测冷冻羊肉卷中羊肉含量。方法：以羊线粒体细胞色素B基因为羊特异性基因,线粒体12S rRNA为内参基因,应用ΔCt法对羊肉含量进行相对定量,并与称重法进行比较,分析基因拷贝数和羊肉脂肪含量对定量结果的影响。结果：荧光PCR法建立的标准曲线在羊肉含量为1%～100%时具有良好的线性关系（R2=0.990 1）。检测7 个市售冷冻羊肉卷样品,荧光PCR方法检测出的羊肉含量与称重法羊肉含量的相关系数为0.945 0（P＜0.001）,平均绝对差异为－11.0%,95%可信区间为（－7.4%,－14.6%）。基因拷贝数不同和羊肉脂肪含量不同对内参基因扩增Ct值影响没有统计学意义（P值分别为0.072和0.206）。结论：荧光PCR法可用于快速检测冷冻羊肉卷中羊肉成分的相对定量。     

一种5 重real-time PCR筛查转基因水稻方法的建立

以转基因水稻中最常用的CaMV35S启动子、NOS终止子、Cry1Ab/Ac基因、HPT基因及SPS水稻内标基因为研究对象,利用5 种不同的荧光信号（FAM、HEX、Taxas Red、Cy5、Cy5.5）进行多重实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）检测方法的研究。通过引物组合筛选、反应体系优化、特异性测试、灵敏度测试、适用性测试等一系列实验,建立了5 重real-time PCR方法,灵敏度可达0.032%。此方法具有灵敏度高、结果准确、通量大等优点,可实现水稻中转基因成分的快速、高效检测。     

产气荚膜梭菌实时荧光PCR和实时荧光RPA检测方法的建立和比较

根据产气荚膜梭菌（Clostridium perfringens）高度保守的plc基因,设计特异性引物和探针,建立产气荚膜梭菌的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）和实时荧光聚合酶重组酶扩增（recombinase polymerase amplification,RPA）检测方法。特异性分析结果表明,建立的两种检测方法特异性强,仅对产气荚膜梭菌有特异性扩增,对其他细菌均无扩增;两种方法的检出限均为1.3 pg/μL。在人工污染模拟样品的检测中,两种方法的检出限均为1.0×102 CFU/mL;实时荧光RPA需要3～13 min即可实现对所有阳性样品的检测,而实时荧光PCR则需要24～46 min（Ct值为17.45～33.65）。实时荧光RPA方法在检测时间、操作方便性和仪器便携性方面,明显优于实时荧光PCR方法。本研究建立的实时荧光PCR和实时荧光RPA检测方法特异性强、灵敏性高、操作方便,为实验设备装备较差的基层检测实验室和疫情突发现场产气荚膜梭菌的快速检测提供有效技术手段。     

原料乳中嗜冷菌计数及产脂肪酶特性的研究

本文主要对乳中的嗜冷菌数的检测方法进行了初步研究。同时,考虑到嗜冷菌能在乳中产生大量的耐热性脂肪酶,力求在脂肪酶活与菌数之间建立联系。即对不同时间测得的酶活与菌数之间的关系进行初步探讨。     

生鲜牛乳总微生物基因组DNA的提取

目的 建立高效快速提取生鲜牛乳中总微生物基因组DNA的方法。方法 以生鲜牛乳为原料, 采用SDS-蛋白酶K法裂解细胞, 酚氯仿有机抽提去除蛋白和醋酸钾溶液沉淀蛋白, 制备样品中总微生物基因组DNA。结果 以NET(Tris?HCl, EDTA, NaCl)作为裂解缓冲液, 蛋白酶K消化得到的基因组DNA纯度和产量较高, 耗时较短; 缓冲液选择NCT(NaCl, CaCl2, Tris?HCl)时, PCR产物特异性低于前者且产量较低。RNA酶消化对产品纯度影响不大且会降低产量。用醋酸钾(KAc)沉淀去除蛋白, 操作快速简单, 耗时最短, 但DNA产量最低。结论 SDS-蛋白酶K法提取的生鲜乳微生物总DNA可以作为牛乳样品进一步检测的分子基础。     

A longitudinal field study to evaluate the diagnostic properties of a quantitative real-time polymerase chain reaction-based assay to detect Staphylococcus aureus in milk

Bacteriological culture as a diagnostic tool for chronic infections with Staphylococcus aureus intramammary infection is not completely satisfactory. The cyclical shedding pattern of Staph. aureus with intervals of low excretion is considered to be the main reason. We recently developed a novel assay for Staph. aureus in milk, based on real-time quantitative PCR (QPCR). In a longitudinal study of chronically infected cows we evaluated the diagnostic properties of this test under field conditions. Diagnostic sensitivity of the novel test proved to be very high with a value of 99.4%; diagnostic specificity was 97.1%. In addition, the shedding patterns of Staph. aureus for the sampling period were analyzed. Using log₁₀-transformed QPCR data and plotting them across sampling time revealed a sinusoidal shedding pattern in 6 of 11 naturally infected quarters. Shedding patterns obtained by QPCR and by bacteriological culture were synchronous. In conclusion, the novel test has a very high diagnostic sensitivity and specificity so that quarters chronically infected with Staph. aureus are reliably detected, independent from their actual shedding quantity.