Details
Store in the dark at -20℃, transport at 2-8℃, valid for one year. Avoid repeated freezing and thawing.
Kit features:
1, specific Sexual identificationCandidatusMycoplasma haemominutum, no cross-reactivity with other organism genomes.
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, it can reduce the contamination of residual DNA.
It is currently known to be parasitic on There are three types of mycoplasma in cat blood: Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis. Before the advent of PCR technology, these three species of mycoplasma were collectively known as Hemobartonella felis, and were later reclassified into the genus Mycoplasma. It has been reported that approximately 15% of cats are carriers. Candidatus Mycoplasma haemominutum parasitizes on the surface of cat red blood cells. It has the weakest pathogenic ability among the above three mycoplasma species and often does not show symptoms. According to different reports,Its infection proportion ranges from 9.6-66.6%, with the highest positive rate among the above three mycoplasma species, and mixed infections are often present.
Commonly used detection methods include microscopy and PCR. The microscopy method has low sensitivity and large experimental errors. It cannot distinguish mycoplasma types and is susceptible to interference from other substances in the blood and artificial smear methods. Mycoplasma is easy to fall off from the surface of red blood cells, resulting in missed detection and may not be detected in chronic attacks. 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. Accurately identifies Candidatus Mycoplasma haemominutum without cross-reaction with the genomes of other organisms. 320 cat blood samples were tested and 133 positive results were obtained, 47 of which were multiple infections. The amplified product was sequenced and confirmed to be Candidatus Mycoplasma haemominutum-specific amplification.
This kit contains hot-start Taq enzyme, dNTP, primers, positive controls, 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 specific monoclonal antibodies. Polymerase activity is inhibited by antibodies 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.Under irradiation with 497nm excitation light, 520nm emission light is produced. SYBR Green I that is not bound to double-stranded DNA produces almost no fluorescence. Therefore, the amount of DNA double strands in the solution can be judged based on the fluorescence value, which can be 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. The Tm value of the target amplification product of this kit is in the range of 81-86 degrees.
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. Provides 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Candidatus Mycoplasma haemominutum，与其他生物基因组无交叉反应。
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。
目前已知寄生于猫血中的支原体有三种：猫血支原体（Mycoplasma haemofelis），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(Hemobartonella felis)，后重新归类为支原体属。有报道大约15%的猫为带菌者。Candidatus Mycoplasma haemominutum寄生于猫红细胞表面，在上述三种支原体中致病能力最弱，常不显现症状，根据不同的报道，其感染比例在9.6-66.6%，在上述三种支原体中阳性率最高，且常有混合感染。
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，在慢性发作时可能无法检测到。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16S rRNA基因的保守区域设计特异性引物，可准确识别Candidatus Mycoplasma haemominutum，与其他生物的基因组无交叉反应。对320个猫血样进行检测，获得133个阳性，其中47个为多重感染。对扩增产物进行测序，证实为Candidatus Mycoplasma haemominutum特异性扩增。
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。本试剂盒目标扩增产物的Tm值在81-86度范围内。
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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