（接上期）

3. Alternative sampling and conservation methods临床采样和保存方法

Oral fluid唾液

In some countries, oral fluid became a widely used sample to detect of PRRS virus genome by RT-PCR and antibodies by ELISA. 使用唾液样本进行PRRSV的核酸和抗体检测已经在一些国家被广泛使用。

There are commercial kits and in-house assays for both techniques. Nonetheless, it is important to remark that due to physical characteristics of oral fluid the RT-PCR and the ELISA kits used for serum analysis cannot be directly applied to this type of sample; the assays must be adapted. 针对PRRSV唾液检测，尽管已经有了商品化和内部测试用的检测试剂盒，但是由于口腔液的物理特性，在进行RT-PCR和ELISA检测之前需要对样品进行前期的处理。

The main advantages of sampling oral fluid in animals, compared to blood collection are:唾液样本与血清样本检测相比优势如下：

·Natural behavior of pigs facilitate sampling. 咀嚼是猪的一种自然习性，便于现场采集

·Not invasive. Collection without restraining animals result in a vast stress reduction. 采样时猪只不需要绑定，减少采样造成的应激

·Increased safety for people. 人员操作更安全

·Reduction in labor/time. 节省时间

·Possibility of testing a large number of individuals in aggregate samples (useful for monitoring purposes). 大面积样本采集更容易进行，有利于监测

Regarding RT-PCR, serum and oral fluid could be considered equivalent during the viraemia period. Serum contains equal or higher concentration of virus than oral fluid for the first two weeks, while it is higher in oral fluid from three-four weeks onwards.病毒血症时期，唾液和血清中的病毒含量差不多；在猪感染后的头2周，血清中的病毒含量稍高于唾液或者差不多；感染后3-4周，唾液中病毒含量要高于血清。

Since excretion period of PRRS virus is usually longer than viraemia, positive results in oral fluid can be obtained during days or even weeks after the end of viraemia. 由于PRRSV排毒的时间要比病毒血症的时间长，因此，病毒血症消失后很多天甚至几周，仍然可以从唾液中检测到病毒。

As a result, pigs could show negative RT-PCR results in serum being positive for oral fluid. During this period, the detection of the virus in oral fluid is usually intermittent. 基于上述原因，当使用RT-PCR进行PRRSV病原检测时，可能会出现血清检测为阳性，但是唾液检测为阴性的情况；在出现这种情况时，通常只能间歇性从唾液中检测到PRSSV。

Another important point to take into consideration is that excretion period is lower and much more intermittent in vaccinated pigs. Obviously, this fact has direct implications in oral fluid diagnostics in a PRRS virus-endemic farm where animals are vaccinated. 另一个重要因素，使用疫苗免疫过的猪只感染PRRSV后其排毒间歇期要比排毒期长。很明显，对于免疫猪群而言，这会影响到唾液检测的效果。

Regarding ELISAs for sera , they could be considered specific with a varying degree of sensitivity; however, in the case of oral fluids, it seems that ELISAs may have a problem of specificity, detecting 5-7% of false positives. 对PRRSV进行血清ELISA检测时，我们考虑的是敏感性问题，而使用唾液样本进行ELISA检测时则要考虑特异性问题，唾液检测中可能会出现5-7%的假阳性。

Conditions during the collection, shipment, storage, etc. of the oral fluid may influence the efficiency of the laboratorial diagnosis: 唾液的采集、运输和储存条件可能会影响实验室诊断的效果：（还有以下影响因素）

·Number of ropes (distribution, size and density of pens). 棉绳的数量（采样分布、猪群大小和密度）

·Material of ropes. Untreated cotton is recommended. 棉绳材质，建议用未处理的棉绳（指的是漂白等处理手段）

·Sick animals interact less, or even do not interact with the rope. 患病猪只精神不佳，不咬棉绳

·Age of animals. Difficulty during sampling of young animals (3-5 weeks-old). 3-5周龄的仔猪不好做唾液样本采集

·Contamination by feedstuff or feces. 粪便和饲料对采样的污染

·Storage conditions and shipment. It is important to note that RNA is easily degraded in biological fluids. Therefore, shipment and storage conditions are critical. 储存条件，PRRSV核酸为RNA，容易降解，运输和储存很重要

·RNA extraction. 病毒RNA提取

Umbilical cord脐带血

When sampling of newborn piglets is mandatory (for example, to determine if vertical transmission exists) several problems appear: to obtain oral fluid is complicated, or even impossible, and with bleeding insufficient or bad quality (coagulation) samples are often obtained. 如果想对新生仔猪进行强制采样（为了观察是否存在垂直传播），可能会碰到以下问题：1）很难或者几乎不可能获得唾液样品；2）采集的血液不足或者质量差（溶血，凝固等）。

In addition, bleeding small animals is a time-consuming task and in some cases die. An alternative to bleed newborn piglets is to collect the fresh umbilical cord. 对新生仔猪采血很费时，而且可能会导致仔猪死亡，因此新鲜脐带可以替代采血用于PRRSV检测。

Advantages of umbilical cord to determine vertical transmission: 脐带检测的优点

·Very easy to collect. It can be carried out directly by farm workers. The umbilical cord must be clamped and cut (portions ≥ 5 cm are recommended).很容易收集，普通饲养员就可以操作，（采集脐带长度＞5cm）

·It can be frozen. 样品可以冷冻保存

·Sampling does not consume too much time. 节省时间

·Sensitivity is as high as bleeding. 检测的敏感性同血清样品一样

As cross-contamination can exist, umbilical cord should be used to determine the status of the litter more than the prevalence of the individual. 脐带样本存在交叉污染的问题，因此，强烈建议以群体（比如窝）为单位进行检测，而不适用于个体检测。

Dried-blood filter papers干燥的血液滤纸（比如FTA卡）

Often, storage conditions of samples in the field are less than optimal, especially when RNA pathogens want to be analyzed; to prevent RNA degradation samples should be refrigerated or frozen. 通常，样品的储存条件都不是很理想，因此，对于容易降解的RNA样本更加要注意保存条件，必须冷冻和冷藏保存。

Dried-blood filter papers are treated filters containing chemicals that lyse cells, denature proteins and protect DNA and RNA from nucleases, oxidation and UV damage. 经过特殊化学处理的干燥血液采集滤纸可以将样品中的细胞溶解，同时使蛋白质变性，保护DNA和RNA免受核酸酶、氧化物和紫外线的损伤。

They can be used to collect, transport and storage up to 125 µL of blood, oral fluid or any other fluid of interest at ambient temperature. 血液，唾液或者其他液体（收集125ul即可）样品都可以使用这种处理过的滤纸来收集，收集的样品可以在常温条件下保存。

Thus, one of the main advantages of this method is that samples can be submitted to the lab by regular post. Samples can be storage for long periods (from months to years) since nucleic acids are immobilized and preserved in the fibers of the matrix. FTA卡好处之一是邮寄方便，信件邮寄即，无须冷藏或者其他包装，另外，由于DNA和RNA已经被固定在基质纤维中，因此可以长期保存（几个月至几年）。

When sensitivity and specificity of PRRS virus real time RT-PCR performed directly in fresh samples or in samples embedded in dried-blood filter papers are compared, it seems that there are some problems detecting the virus in embedded oral fluids. 比较新鲜样品和干的血滤纸固定的样品进行PRRSV实时定量荧光RT-PCR的敏感性和特异性发现，固定的唾液样品在进行PRRSV检测存在着一些问题。

Regarding embedded blood, the sensitivity could decrease due to the low amount of sample collected and in those samples with low quantity of virus. 由于滤纸可以收集的样品很少，就会导致病毒含量低，从而降低检测的敏感性。

Thus, fresh samples with RT-PCR values near the cut-off (high Ct, it means low quantity of virus) could result negative when samples are transferred to the dried-blood filter paper. As a result, it seems that the method could loss sensitivity in animals not acutely infected. 因此，当新鲜样品检测的拷贝数与CUT-OFF值接近时（高CT值，病毒含量低），使用滤纸收集的样品检测结果可能为阴性，说明这种方法处理样本的敏感性低，不能准确的反应感染情况。

（未完待续）

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