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.     

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.     

Single-molecule real-time sequencing reveals differences in bacterial diversity in raw milk in different regions and seasons in China

The quality of raw milk is a key factor influencing the whole dairy processing chain. The richness and diversity of bacteria in raw milk affect its quality and safety. However, traditional microbial detection methods mainly depend on the known microbe culture and are often time consuming. Thus, the development of efficient ways for supervising any possible microbiological contamination is desiderated. In the current work, single-molecule real-time (SMRT) sequencing, developed by Pacific Biosciences (PacBio), was applied to acquire long reads and applied for discrimination of bacteria at species level. Forty samples of raw milk obtained from Beijing, Hebei, Inner Mongolia, Shanghai, and Guangdong in China during summer, autumn, and winter were investigated. Among 35 bacteria species identified in these samples, Acinetobacter albensis, Pseudomonas gessardii, Pseudomonas weihenstephanensis, and Rahnella inusitata were the bacteria with the highest relative abundance in the overall sample, whereas the bacteria with the highest relative abundance in raw milk samples of different origins and seasons are different. Significant differences in bacterial richness and bacterial community diversity in raw milk grouped according to different production areas and different sampling seasons were confirmed by Welch's t-test. Interestingly, the transport distance and transport time positively correlated with the relative abundance of Pseudomonas weihenstephanensis, suggesting that the content of this bacteria was expected to be a standard for evaluating the freshness of raw milk. Pathogens Bacillus cereus and Klebsiella pneumoniae were detected in most samples, indicating that the raw milk was at risk of contamination by pathogenic bacteria. Moreover, the findings of this study provide important evidence for quality and safety monitoring and biological control of raw milk.     

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.     

Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 μL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.     

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.     

乳中蛋白酶与UHT乳贮存中的胶凝现象

乳中的蛋白酶有2个主要的来源途径，乳中天然存在的蛋白酶和由某些微生物产生的蛋白酶，其中纤维蛋白溶酶和嗜冷菌产生的耐热性蛋白酶是存在于UHT乳中的主要蛋白酶，这些蛋白酶非常耐热，经UHT灭菌处理仍可存活。耐热性蛋白酶在UHT乳贮存中水解乳蛋白质从而导致了UHT乳的胶凝．简要介绍了纤维蛋白溶酶、嗜冷菌耐热性蛋白酶的性质、活性测定方法，论述了这些酶引起的UHT乳的胶凝性质、形成原因及影响因素，并提出了一些防止措施。     

聚合酶链式反应技术快速检测原料乳中荧光假单胞菌

目的 利用PCR（polymerase chain reaction）技术建立一种快速、准确检测原料乳中最常见有害嗜冷微生物——荧光假单胞菌的方法。方法 以荧光假单胞菌蛋白酶基因aprX为检测靶标,设计特异性PCR简并引物,建立原料乳中荧光假单胞菌PCR检测体系,对该体系的特异性及检测限进行评价。结果 筛选到特异性引物F3：5’-WSNGGNGGNGAYTTYCAYATGAC-3’;R3：5’-RTCRTTNCCNCCNCCRTCCC-3’,建立了最佳PCR检测体系：引物浓度0.5 μmol/L、Taq DNA聚合酶添加量0.4 μL、dNTPs浓度0.16 mmol/L、Mg²⁺浓度1.6 mmol/L,退火温度56.4 ℃,扩增35个循环。此检测方法对荧光假单胞菌具有特异性,检测限为2.57×10³ CFU/mL。结论 本方法操作简便,特异性强,检测限低,对快速检测原料乳中嗜冷菌,保障原料乳品质与安全具有一定参考意义。     

原料乳中优势嗜冷菌株的确定及其微生物学特征研究

对原料乳样品进行嗜冷菌的分离筛选,结果得到4株优势嗜冷菌。本文研究了这4株优势嗜冷菌的生长特性及产脂肪酶特性,分别得到了4株优势嗜冷菌株的最佳生长条件及最佳产脂肪酶条件。      

Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique

A polymerase chain reaction (PCR) assay was developed for the specific identification of cows' milk in sheep's and goats' milk by using primers targeting the mitochondrial 12S rRNA gene. The use of a forward primer complementary to a conserved DNA sequence, along with a reverse primer specific for cow, yielded a 223-bp fragment from cows' milk DNA, whereas no amplification signal was obtained in sheep's and goats' milk DNA. The technique was applied to raw, pasteurized, and sterilized milk binary mixtures of cow-sheep and cow-goat, enabling the specific detection of cows' milk with a good sensitivity threshold (0.1%). The proposed PCR assay represents a rapid and straightforward method applicable to the authentication of milk and other dairy products in routine analysis.     

食品中荧光假单胞菌聚合酶链式反应检测体系的建立和评价

建立用聚合酶链式反应(polymerase chain reaction,PCR)检测食品中荧光假单胞菌的方法。分别针对16～23S rRNA基因间隔区序列、gyrB基因以及通过生物信息学方法发掘到的4个种特异性基因设计6对检测引物,通过初步特异性实验,筛选出一对种特异性最佳的引物。最终建立以gyrB基因为检测靶点的PCR扩增体系,并对体系进行系统评价。结果表明:该方法可特异检测荧光假单胞菌的存在,纯DNA检测灵敏度为14.9fg/μL(2～3拷贝/μL),纯培养物检测灵敏度为2.8×102CFU/mL。豆奶样品经15h充分增菌可提高检测灵敏度至0.28CFU/25g。     

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。     

原料乳中嗜冷假单胞菌危害及控制研究进展

假单胞菌是对原料乳危害最大的嗜冷菌之一,极易在冷藏条件下成为优势菌群。大多数嗜冷假单胞菌有分泌蛋白酶和脂肪酶的能力,并导致原料乳及产品变质。本文对原料乳假单胞菌的危害特点,快速检测和新型控制工艺研究进展进行综述。最后探讨了嗜冷假单胞菌的研究方向。      

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.     

Dynamics of relationship between the presence of Coxiella burnetii DNA, antibodies, and intrinsic variables in cow milk and bulk tank milk from Danish dairy cattle

Milk samples of 12 Danish dairy herds were collected 3 times during an 11-mo period and tested for Coxiella burnetii DNA by real-time PCR, detecting the IS1111 element, and for the presence of antibodies against the bacterium by ELISA. On average, 25% of 1,514 samples were seropositive and 32% were positive for C. burnetii DNA. Among the 485 DNA-positive samples, quantification cycle values ranging from 15.8 to 37.8 were found. Test sensitivity did not increase after DNA extraction from the cream fraction compared with full milk. The relationship between antibody levels and bacterial shedding was investigated among 166 cows from 9 herds. The prevalence levels of C. burnetii DNA and antibodies in the herds were found to be rather stable for 6 of the herds. The test results were highly influenced by results obtained 3 to 7 mo earlier. A significant association between the antibody titer and the DNA shedding level at the same and the preceding visit was found. In addition, a significant association between the antibody titer and the antibody titers 3 to 11 mo earlier was found. A multivariable analysis identified a significant increase in C. burnetii DNA shedding with increasing parity and increasing protein concentration in milk. The antibody levels in bulk tank milk and prevalence levels of C. burnetii DNA and antibodies in individual cow milk samples were correlated. A significant correlation was also found between the quantification cycle values of the cow samples (weighted according to milk yield) and the C. burnetii concentration in bulk tank milk.     

Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique

Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.     

Growth of a rifampicin-resistant strain of a proteolytic psychrotroph,Pseudomonas fluorescens,in raw and ultra heat-treated milk

Pseudomonas fluorescens UQM2490, resistant to 250 μg rifampicin/ml, was derived from P. fluorescens JC1, a proteolytic psychrotroph isolated from raw milk. Growth of UQM2490 was followed in raw and ultra heat-treated milk, by viable counting on rifampicin-containing agar medium. The growth curves obtained demonstrate slower growth in raw milk than in treated milk and the variation in growth with change in inoculum level. Generation times in ultra heat-treated milk ranged from 9.5 to 14.1 h compared with 9.6 to 33 h in raw milk.