Typing method for detection of HCMV gBn by real-time fluorescent quantitative PCR

A technology of real-time fluorescence quantification and typing method, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of inability to quantify DNA, etc., and achieve the effect of avoiding human judgment, intuitive results, and a wide range of quantification

Inactive Publication Date: 2009-04-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the technologies used for HCMV gB typing are restriction length polymorphism analysis and qualitative PCR, which can type HCMV gB, but cannot quantify its DNA

Method used

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  • Typing method for detection of HCMV gBn by real-time fluorescent quantitative PCR
  • Typing method for detection of HCMV gBn by real-time fluorescent quantitative PCR
  • Typing method for detection of HCMV gBn by real-time fluorescent quantitative PCR

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Experimental program
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Effect test

Embodiment 1

[0035] Equipment: PCR instrument SLAN (Shanghai, Hongshi), UV spectrophotometer HP8453 (HP company, Germany), gel image analysis system BioSenSC300 (Shanghai Shanfu Scientific Instrument Co., Ltd.), low-temperature high-speed centrifuge (Biofuge 28RS) (Heraeus company, Germany), constant temperature water bath box (Model DK-8D (Shanghai Senxin Experimental Instrument Co., Ltd.).

[0036] Reagents: TAQ enzyme (Promega, USA), nucleic acid extract (Shanghai Zhijiang Biology), primers (Invitrogen, USA). Specific ratio of nucleic acid lysate: 10mmol / L Tris-cl (pH8.0), 0.1mol / L EDTA (pH8.0), 20ug / ml Trypsin, 0.5% SDS.

[0037] PCR Primers and Probes

[0038] HCMV-gB1-1:5'-cggttcagtctctcaacg-3'

[0039] HCMV-gB1-2: 5'-caccacatctccgtacttg-3'

[0040] Probe HCMV-gB1-3: FAM-ttcccaaacggtcagc-BHQ1-3;

[0041] HCMV-gB2-1: 5'-ctcgtacgacgtcgtctcaa-3'

[0042] HCMV-gB2-2: 5'-cacacgcgataggggtactt-3'

[0043] Probe HCMV-gB2-3: 5'-FAM-gagaatagactgac-BHQ1-3';

[0044] HCMV-gB3-1: 5'-ctcgta...

Embodiment 2

[0077] According to the above experimental procedures, 1207 samples were selected for real-time fluorescence quantitative PCR to determine the genotypes of HCMVgBn1, 2, and 3 after transplantation recipients, including 40 liver transplant recipients with 366 specimens, and 120 kidney transplant recipients with 350 specimens , 55 cases of bone marrow transplant recipients, 481 samples, the results showed that the positive rate of gBn1 type was 5.3%, the positive rate of type 2 was 1.0%, the positive rate of type 3 was 2.7%, and type 4 was not detected; The difference (p>0.05), the mixed infection of gBn1 and type 3 accounted for 42.3% of gBn1 positive; the distribution of different transplant types in the positive samples had no difference. Due to the quantification of HCMV gB, it can not only detect the infection status of HCMV, but also judge the prognosis of patients. For example, the prognosis of type 1 infection is generally better; mixed infection has higher viral load and...

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Abstract

The invention discloses a method for detecting HCMV gBn classification by real-time fluorescence quantitative PCR. The method comprises the following steps: a fluorescent probe is added in a PCR reaction system, the PCR process is monitored in real time by the fluorescent signal accumulation and quantitative and qualitative analysis is carried out to determine the type of HCMV gBn. The invention is closed reaction without PCR post treatment, avoids pollution, has strong specificity and high sensitivity, adopts logarithmic phase analysis and abandons destination data and has accurate quantification; the real-time fluorescence quantitative PCR technology can carry out classification and quantification on HCMV gB; the on-line real-time detection by instruments has intuitive result and avoids artificial judgment; double-detection or multi-detection by one tube can be realized; and the operation is safe and the time is shortened. The common PCR needs about 1 to 2 days, while the method of the invention generally needs only 3 to 4 hours, thus improving the efficiency; the invention can be used for clinically detecting gBn classification of HCMV-infected crowd, predicting the prognosis, observing HCMV activity infection and guiding clinical treatment.

Description

technical field [0001] The invention relates to gene amplification and detection, in particular to a gBn type detection method of human cytomegalovirus. Background technique [0002] Human cytomegalovirus (HCMV) is a common infection in the population. Most people with primary infection are asymptomatic and become asymptomatic carriers, but if immunodeficiency or immunosuppression can cause serious consequences, such as leukopenia, pneumonia, gastrointestinal disease, weakening and rejection of graft function, encephalitis, retina Inflammation, etc.; in terms of prenatal and postnatal care, if intrauterine HCMV infection can cause serious sequelae in the baby. [0003] gB is one of the most important envelope glycoproteins of HCMV. It is the most immunogenic among the proteins encoded by HCMV. It is the key target of neutralizing antibodies and participates in cellular responses. It is a potential HCMV virulence factor. HCMV subtypes are usually classified as gB types. Cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 李兰娟马伟杭范骏张旋陈晓明杨美芳高海女赵宏胡建华
Owner ZHEJIANG UNIV
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