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Reverse transcription - polymerase chain reaction of hepatitis delta virus infection in the Application and Significance

4.5td, 2.5mMdNT4.5td, 1O00PM/glRandomHex0.5td (or HD4 primer 3td), 0.1MDTTlol, 40u/glRNasin0.5td,[link widoczny dla zalogowanych], 200u/glM-MLV enzyme lgL, plus H2o to 35; plus serum or liver tissue extracts l0l,[link widoczny dla zalogowanych], set 37 ℃ 90rain. Followed by 98 ℃ 5rain inactivation. (2) The first round of PCR amplification: the RT reaction tube by adding 10 × Taq buffer 05td. HD outside of each primer, 2td, TaqDNA enzyme 3u. PCR cycle program: 95 ℃ 30s,[link widoczny dla zalogowanych], 55 ℃ lmin, 72 ℃ 2min, 40 cycles, then 72 ) material within the I, Taq enzyme and the first round PCR product 2td. PCR cycle with the former. Take lO, ul second round PCR products were 2% agarose gel (with EB) electrophoresis, observed in the UV light. To PBR322 / H; nfT hydrolysates as control, appears consistent with the positive control amplified 366bp band as positive. 1-4 Statistical data processing by testing. 2 Results 2.1 RT-PcR Conditions for Determination of HDVRNA results (1) lO patients with HDV-M-positive serum. Using random primers and specific primers (HOD for RT compared by 2 PCR amplification, 5 cases were positive for the former, the latter 2 cases were positive,[link widoczny dla zalogowanych], random primer method is better than specific primers. But the specific primers using simple, inexpensive , adjusted conditions,[link widoczny dla zalogowanych], part of the test is still used. (2) 10 patients with positive serum HDV-M, the use of 10 × Taq buffer with 15raM 25raM MgCI2 concentration compared amplification by PCR were determined after 2, 5 cases of the former positive, which is only 1 positive, so the MgC12 concentration used 25raM. 2'2 Viral Hepatitis HDVRNA the fish out rate shown in Table 1, 98 cases of serum. HDV-M HDV positive group of Chinese Physician 1999 on June 6 Volume 1 RNA detection rate was 56.6% (30/53), CAH and LC with the highest case detection rate (respectively 55.9% and 62.6%); HDV-M negative rate was 8.9% (4 / 45); 10 healthy blood donors were negative determination. Table 1. various types of viral hepatitis patients with RT - PCR detection rate of diagnostic determination HDVRNA last * two comparison: P <001 serum HDV-M relationship with HDVRNA results: HDAg-positive serum HDVRNA positive rate 45.5% (5 / 11) HDAg / positive serum anti HDIgM were 60.0% (18/30), anti- HDIgM was positive in 62.5% (5 / 8), positive anti-HI3 50.0 (2 / 4). because of the small number of patients. have not been statistically. 2.3 Determination of 9 cases of liver HDVRNA HDV-M-positive cases application of liver tissue determined HDVRNA RT_ a PcR positive in 6 cases (see photo). HDVcDNA digoxigenin-labeled probe in situ hybridization method to measure HDVRNA also positive in 6 cases, the sensitivity of both methods broadly in line; 2 cases HDv-M-negative liver tissue were negative. are two ways to co-positive in 5 cases. the common negative in 4 cases, in line with rate of 81,8%. rPcR Law Wai tied with measured liver HI) VRNA Kou Results 1. To Markee (pBR322/HinfL): 2. HDV positive control serum; 366bp band went to see fluorescence; 3. Negative control: 4.5.67 4 cases of liver Qi is not the liver tissue, HDVRNA bolt left positive. 366bto fluorescence band can be seen at the 8-state non-liver competition for the】 sick liver maggots Kun, HDVRNA bolt stripping limiting, 366bp band 3 fluorescence at no discussion the first time in 1986 Wang_6 cloned from the serum of infected chimpanzees and sequence determination of the HDVcDNA. In recent years, domestic and international applications of RT-PCR technique has been successively reported HDVRNA 1 - 3], we refer to domestic and international experience, the establishment of PT - PCR method for clinical determination of serum and liver tissue HDVRNA main steps: (1) Select HDV relatively conservative area as the target gene primers; (2) random primers or specific primers for reverse transcription; (3) selection of M-MLV reverse transcriptase; (4) appropriate MgC