Store in the dark at -20℃, transport at 2-8℃, valid for one year. Avoid repeated freezing and thawing.

 

 

 

Kit features:

1. Specific recognition of Mycoplasma felis and Mycoplasma canis without interference from other parasitic microorganisms of cats and dogs.

2. Use hot-start Taq enzyme to inhibit non-specific amplification and reduce background fluorescence.

3, with positive control sample, which can be used to test the effectiveness of the kit and verify the sample to be tested Does it contain PCR inhibitors?

4, with ROX reference dye to help correct sampling errors and differences between tubes.

5, with UDG enzyme and dUTP, which can reduce the contamination of residual DNA.

 

Mycoplasma haemofelis , also known as Mycoplasma felis and Eperythrocytosis felis, it parasitizes on the surface of cat red blood cells and is pleomorphic. It can be spherical, ring-shaped or rod-shaped. It has a destructive effect on red blood cells and can cause infectious hemolytic anemia. Sometimes it is an acute attack, which can be fatal; sometimes it is a chronic attack, with symptoms such as lethargy, anorexia, pale mucous membranes, jaundice, fever, and splenomegaly, and may occur repeatedly. The route of transmission is through blood, including bites from blood-sucking insects, bites, blood transfusions, etc.


There are currently three known mycoplasmas that parasitize in cat blood: Mycoplasma felis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis; before the advent of PCR technology, these three mycoplasmas were collectively called Haemobartonella felis. It has been reported that about 14% of anemic cats are positive for blood mycoplasma. The pathogenic ability of Mycoplasma felis is stronger than the other two mycoplasmas in cat blood.


There are two types of mycoplasma that parasitize in dog blood: Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. Mycoplasma canis and Mycoplasma felis are very closely related, have similar morphologies, and their gene sequences are almost 100% identical. Cats can be infected with Mycoplasma canis, but dogs cannot be infected with Mycoplasma felis. Mycoplasma canis infection sometimes does not show symptoms, and sometimes it may cause some symptoms, including: dehydration, mild anemia, tachycardia, jaundice, and death, but it rarely causes obvious hemolysis unless splenectomy is performed or an infection occurs. immunosuppressed. Mycoplasma canis is the most common hemophilic mycoplasma in dogs and is extremely widely distributed, with a positive rate of approximately 0.5-40%.

Haemophilic MycoplasmaCommonly used detection methods include microscopy and PCR. The microscopy method has low sensitivity and large experimental errors. It cannot distinguish the types of mycoplasma and is easily interfered by other substances in the blood and artificial smear methods. Mycoplasma can easily fall off the surface of red blood cells, resulting in missed detection. The PCR method has obvious advantages in sensitivity and can distinguish mycoplasma species. Compared with the ordinary PCR method, the quantitative PCR method can not only accurately quantify, but also be more convenient to operate and less affected by environmental pollution.


This kit designs specific primers for the conserved region of the 16S rRNA gene of Mycoplasma felis and can accurately identify Mycoplasma felis and Mycoplasma canis cross-reacts only with Candidatus Mycoplasma haemobos. Candidatus Mycoplasma haemobos is a parasite in cattle blood and will not affect the detection of this kit. Testing of 320 cats showed that the overall positive rate for Mycoplasma hemophilum was 43.4%, and the positive rate for Mycoplasma felis was 12.8%.


This kit contains hot-start Taq enzyme, dNTP, primers, positive control, SYBR Green I dye, ROX dye, and UDG to prevent residual DNA contamination ), users only need to prepare DNA samples to use.



Hot-start Taq enzyme combines DNA polymerase with a specific monoclonal antibody, and the polymerase activity is inhibited by the antibody at room temperature. During the denaturation stage of the PCR cycle, the antibody is removed by heat and the polymerase activity is restored, thus obtaining a "hot start" function, which can reduce the contamination of residual DNA and improve PCR amplification efficiency, sensitivity and target fragment yield.


SYBR Green I can bind to the minor groove of DNA double strands, and after binding, it produces 520nm emission light under irradiation of 497nm excitation light. SYBR Green I that is not bound to double-stranded DNA produces almost no fluorescence. Therefore, the D in the solution can be judged based on the fluorescence value.The amount of NA double strands is used to detect the accumulation of products during the PCR reaction in real time. The higher the initial template concentration and the higher the reaction cycle number, the higher the double-stranded DNA content and the higher the fluorescence value. It is worth noting that SYBR Green I can also bind non-specific PCR products and primer-dimers, and the fluorescence produced in this case is indistinguishable from the target amplification product. To determine whether the fluorescent signal comes from the target amplification product, a melting curve can be drawn. By gradually and slowly raising the temperature, double-stranded DNA is dissociated into single-stranded DNA. At the same time, changes in fluorescence value are detected, a curve is drawn, and the temperature at which the DNA melts is observed based on the fluorescence value.

UDG and dUTP can prevent the amplification of residual DNA from the previous step. dUTP ensures that amplified DNA contains uracil. UDG removes uracil residues from single- or double-stranded DNA, thereby preventing uracil-containing DNA from serving as template for subsequent rounds of PCR. The UDG incubation step before the start of the PCR cycle destroys uracil-containing PCR products. After this contamination removal step, UDG is inactivated by high temperature during the normal PCR cycle and has no impact on the PCR reaction.


ROX does not participate in the PCR reaction, and the fluorescence does not change during the PCR reaction. It can provide a baseline for correcting sampling errors and differences between tubes, allowing the instrument to automatically analyze the ratio of the reported fluorescence to the internal reference ROX fluorescence, making quantification more accurate. The excitation wavelength of ROX dye is 584nm and the emission wavelength is 612nm. After correction with ROX, the experimental error can be reduced by about ten times.

 

 

 

 

 

-20℃避光保存，2-8℃运输，有效期一年。避免反复冻融。

 

 

试剂盒特点：

1， 特异性识别猫血支原体和犬血支原体，不受猫和犬的其他寄生微生物的干扰。

2， 使用热启动的Taq酶，可抑制非特异性扩增，降低背景荧光。

3， 带有阳性对照样品（组分C），可用于检验试剂盒有效性，以及验证待测样品中是否含有PCR抑制剂。

4， 带有ROX参比染料，帮助校正加样误差和管间差异。

5，带有UDG酶和dUTP，可降低残留DNA的污染。

 

猫血支原体（Mycoplasma haemofelis），或称为猫嗜血支原体、猫附红细胞体，寄生于猫红细胞表面，具多形性，可呈现为球状、环状或杆状，对红细胞有破坏作用，可引起传染性溶血贫血症，有时呈急性发作，可致命；有时呈慢性发作，出现嗜睡、厌食、粘膜惨白及黄疸、发烧、脾脏肿大等症状，并可能出现反复发作。其传播途径为血液传播，包括：吸血昆虫叮咬、咬伤、输血等。

 目前已知寄生于猫血中的支原体有三种：猫血支原体，Candidatus Mycoplasma haemominutum和Candidatus Mycoplasma turicensis；在PCR技术出现之前，这三种支原体被统称为猫血巴尔通氏体（Haemobartonella felis）。有报道约14%的贫血猫呈血液支原体阳性。猫血支原体的致病能力强于猫血中的另两种支原体。

 寄生于犬血中的支原体有两种：犬血支原体（Mycoplasma haemocanis)和Candidatus Mycoplasma haematoparvum。犬血支原体与猫血支原体亲缘关系非常近，形态也类似，基因序列几乎100%一致。猫可以感染犬血支原体，而犬不能感染猫血支原体。犬血支原体感染后有时不表现出症状，有时可能引起一些症状，包括：脱水、轻度贫血、心跳过速、黄疸、死亡，但是很少引起明显的溶血，除非是做了脾切除手术或者发生了免疫抑制。犬血支原体是犬类中最常见的嗜血支原体，分布极为广泛，阳性率大约在0.5-40%之间。

 嗜血支原体常用的检测方法包括显微镜镜检法和PCR法。镜检法灵敏度低，实验误差大，无法区分支原体种类，易受血液中其他物质以及人工涂片方法的干扰，且支原体易从红细胞表面脱落，造成漏检。而PCR法在灵敏度上则有明显的优势，且可以区分支原体种类。定量PCR法与普通PCR法相比，不仅可以精确定量，而且操作更为方便，更少受环境污染的影响。

 本试剂盒针对猫血支原体的16S rRNA基因的保守区域，设计特异性引物，可准确识别猫血支原体和犬血支原体,仅与Candidatus Mycoplasma haemobos有交叉反应。Candidatus Mycoplasma haemobos寄生于牛血中，不会对本试剂盒的检测产生影响。对320只猫的检测表明，嗜血支原体的总阳性率为43.4%，猫血支原体的阳性率为12.8%。

 本试剂盒包含热启动Taq酶、dNTP（以UTP代替了TTP）、引物、阳性对照品、SYBR Green I染料、ROX染料，和用以防止残余DNA污染的UDG（尿嘧啶-N-糖苷酶），用户只需准备好DNA样品即可使用。

 热启动Taq酶结合了特异性单克隆抗体的DNA聚合酶，在室温下聚合酶活性被抗体抑制。在PCR循环的变性阶段，抗体受热被去除，聚合酶活性恢复，因此获得“热启动”功能，可减少残余DNA的污染，提高PCR扩增效率、灵敏度和目的片段产量。

 SYBR Green I可以与DNA双链的小沟结合，结合后在497nm激发光照射下产生520nm的发射光。未结合双链DNA的SYBR Green I基本不产生荧光。因此可以根据荧光值的大小判断溶液内DNA双链的多寡，用于实时检测PCR反应过程中产物的累积量。初始模板浓度越高，反应循环数越高，则双链DNA含量越高，荧光值也就越高。值得注意的是，SYBR Green I也可以结合非特异性PCR产物以及引物二聚体，此时产生的荧光与目标扩增产物无法区分。为了判断荧光信号是否来自目标扩增产物，可以绘制熔解曲线。通过逐步缓慢升温，使双链DNA解离为单链DNA，同时检测荧光值的变化，绘制曲线，根据荧光值观察DNA解链的温度。

 UDG和dUTP可以阻止前次步骤残余DNA的扩增。dUTP可以确保扩增后的DNA中均含有尿嘧啶。UDG可从单链或双链DNA上去除尿嘧啶残基，从而阻止含有尿嘧啶的DNA作为下几轮PCR的模板。在PCR循环开始前的UDG孵育步骤，可以破坏带有尿嘧啶的PCR产物。经过该去除污染步骤后，UDG在正常的PCR循环过程中被高温灭活，对PCR反应没有影响。

 ROX不参与PCR反应，在PCR反应过程中荧光没有变化，可以提供一条基线用于校正加样误差和管间差异，便于仪器自动分析报告荧光与内参ROX荧光的比值，使定量更准确。ROX染料的激发波长为584nm，发射波长为612nm。用ROX进行校正后可以使实验误差减小十倍左右。

 

 

 

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