PCR (polymerase chain reaction) kit for detecting Wenckebach eperythrozoon
A technology with erythrocytes and kits, which is applied in the field of genetic testing, can solve the problems of indistinguishability, and achieve the effects of simple operation, high sensitivity, and easy standardization
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Embodiment 1
[0021] Blood samples were collected from dairy cows, cattle and buffaloes infected with Eperythrozoon wennerii, and whole blood genomic DNA was extracted using Promega's DNA extraction kit (E.Z.N.A. Gel Extraction Kit).
[0022] Select the 16S rRNA gene nucleotide sequences of cattle, sheep, pigs, cats, and rabbits included in GenBank, design and synthesize a pair of primers for amplifying the 16S rRNA of Eperythrosomes, the theoretical amplification fragment is 446bp, and the primers The sequence is as follows:
[0023] Upstream primer Cm1: 5'-cgaacgagtggaacttgttctgc-3' (SEQ ID NO.1);
[0024] Downstream primer Cm2: 5'-tagtaccatcaaggcgtgctc-3' (SEQ ID NO.2);
[0025] The primer sequences were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd.
[0026] The prepared genomic DNA was used as a template, and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were used as primers for PCR amplification. The PCR reaction system is shown in Table 1:
[0027] Table 1. Compos...
Embodiment 2
[0032]Cut out the target band in the agarose gel after electrophoresis in Example 1, and recover the target band according to the instructions of Promega's gel extraction kit (E.Z.N.A. Gel Extraction Kit); take the recovered target band and T The vector (pMD18-T) was connected overnight at 16°C, and then transformed into JM109 competent cells. After overnight culture, white colonies were picked and inoculated in 1mL LB liquid medium, and then cultured overnight at 37°C and 150rpm, and identified by PCR Positive clones were selected and sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing. The sequence result of the blood sample genome of cows infected with Eperythrozoon wennerii as a template is shown in SEQ ID NO.3, and its base number is 415bp; the sequencing result of the blood sample genome of cows and buffalo infected with Eperythrocytos wennerii as templates As shown in SEQ ID NO.4 and SEQ ID NO.5 respectively, the number of bases is 416bp. Comparing the obta...
Embodiment 3
[0034] According to the PCR method of Example 1, the blood sample genomic DNA of cows infected with Eperythrozoon wennerii was used as a positive control, and the Eperythrozoon porcine, Eperythrozoon sheep and Escherichia coli DNA were respectively used as templates for PCR detection, and the amplified products were used for % agarose gel electrophoresis detection, the results are as follows figure 2 shown. Depend on figure 2 It can be seen that no band was amplified using Eperythrozoon porcine, Eperythrozoon ovis and E. coli DNA as templates, but a band of about 416 bp was amplified from the blood sample genomic DNA of cows infected with Eperythrozoon wennerii. Show that the primers shown in SEQ ID NO.1 and SEQ ID NO.2 have good specificity, and can specifically amplify the 16S rRNA of Eperythrozoon Wennneri, but cannot amplify Eperythrozoa porcine, Eperythrozoon sheep, etc., so it can It is used for the specific detection of Eperythrozoon Wennneri.
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