Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi · 2005
[Detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma by hemi-nested PCR].
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Jiang Hui-Yong, Zhang San-Quan, Han Xi-Qun, Song Lan-Ying, Zhu Mei-Gang, Zhao Tong
Аннотация
OBJECTIVES: To explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method.
METHODS: bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.
RESULTS: bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.
CONCLUSION: There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.
METHODS: bcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.
RESULTS: bcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.
CONCLUSION: There are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.