器官移植术后的嵌合现象

为研究器官移植术后的嵌合现象，术后利用聚合酶链反应（ＰＣＲ）技术检测３例接受男性供体器官的女性受者外周血及皮肤组织中的Ｙ染色体特异性ＤＮＡ片段。结果在１例小肠移植受者的外周血及皮肤组织中出现Ｙ染色体特异性ＤＮＡ片段；２例肾移植受者的外周血中也出现Ｙ染色体特异性ＤＮＡ片段。表明在器官移植术后存在着供体细胞向受体组织的移行嵌合。认为促进嵌合的出现及保持嵌合的平衡，将有利于防治排斥反应和移植物抗宿主病。     

Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real‐time polymerase chain reaction

Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time‐consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real‐time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast—it permits the scanning of DNA from a patient in one and a half hours—and reliable, minimising the risk of cross‐contamination and false‐positive and false‐negative results.     

多聚酶链反应检测尿中结核杆菌的临床意义

应用多聚酶链反应（ＰＣＲ）对８６例患者（１０例经病理诊断为肾结核，６９例可疑肾结核，７例单纯附睾结核）和３０例健康对照者进行连续２日晨尿结核菌检测。１０例肾结核患者检出均阳性；可疑肾结核者第一次检出９例，第二次为６例；７例附睾结核者两次无一阳性；对照组有１例二次检查均为阳性。认为ＰＣＲ对尿中结核杆菌检出率高、准确、快速，值得在临床上推广应用。     

Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation

BACKGROUND: Evaluation of the outcome of successful bone marrow transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. METHODS: A competitor standard was generated via PCR using a mutagenic primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were analyzed by computing densitometry. The ratio of competitor/target (Y-specific) DNA for each sample pair was calculated. RESULTS: Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calculated to be around 50%. CONCLUSIONS: This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.     

PCR检测非细菌性前列腺炎沙眼衣原体的初步研究

对３０例非细菌性前列腺炎（ＮＢＰ）患者的前列腺按摩液（ＥＰＳ）沙眼衣原体（ＣＴ）进行了聚合酶链反应（ＰＣＲ）检测，并与经二乙氨基葡聚糖处理的ＨｅＬａ细菌培养法进行对比研究，结果发现，６例ＰＣＲ和细胞培养双阳性，２１例ＰＣＲ和细胞培养双阴性，３例两种检测结果不一致，包括２例ＰＣＲ阳性但培养阴性，１例培养阳性但ＰＣＲ阴性，将ＰＣＲ与细胞培养进行比较，ＰＣＲ的敏感性为８５．７％，特异性９１．３％，阳性预     

Rapid sensitive molecular diagnosis of pyogenic spinal infections using methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S ribosomal RNA gene-based universal polymerase chain reaction

Background contextRapid diagnosis and accurate detection of etiological agents in pyogenic spinal infection (PSI) patients are important.PurposeThe purpose of this study was to evaluate the clinical usefulness of methicillin-resistant Staphylococcus-specific polymerase chain reaction (MRS-PCR) and broad-range universal PCR (U-PCR) for diagnosing PSI.Study designA prospective diagnostic study.PatientsThirty-two clinically suspect PSI patients and six control patients who underwent computerized tomography–guided biopsy and/or surgical treatment were enrolled.MethodsTissue samples were examined by microbiological culture, histopathology, and real-time PCR (MRS-PCR and U-PCR). The diagnostic accuracy of real-time PCR was analyzed based on the definitive diagnosis of infection, defined as a positive result from microbiological culture or histopathology.ResultsAll six control subjects were negative for PSI for all analyses. Twelve clinically suspect PSI subjects received definitive diagnoses (PSI group). The non-PSI group consisted of six control subjects plus the remaining 20 patients from the PSI clinically suspect group. MRS-PCR results were positive for all MRS-cultured PSI subjects. U-PCR was positive for all subjects in the PSI group with one discrepancy between real-time PCR and microbiological culture results in differentiation between gram-positive and gram-negative bacteria. In the non-PSI group, MRS-PCR and U-PCR were positive in three and seven cases, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of MRS-PCR for diagnosing MRS infection were 1.00, 0.91, 0.57, and 1.00, respectively; those for the diagnosis of bacterial infection with U-PCR were 1.00, 0.73, 0.63, and 1.00, respectively.ConclusionIdentification of MRS infection and ability to differentiate between gram-positive and gram-negative bacteria is rapidly achieved using MRS-PCR and U-PCR. Real-time PCR provides a sensitive molecular diagnosis of PSI and may contribute to antibiotic selection.     

Detection of fungi in the sinus mucosa using polymerase chain reaction.

OBJECTIVE: To compare the presence of fungi in the sinus mucosa of patients with and without chronic rhinosinusitis. STUDY DESIGN AND SETTING: Prospective observational study using polymerase chain reaction and conventional culture to detect fungi in the sinus mucosa. Middle meatus mucosal samples were collected from 31 patients with chronic rhinosinusitis and 14 control subjects. RESULTS: Fungi were detected in 6.5% of subjects with chronic rhinosinusitis and in none of the control subjects using polymerase chain reaction. Fungi were detected in 29% of subjects with the combination of inhalant allergies, nasal polyposis, and asthma. Fungi were detected in none of the subjects without the combination of these three comorbidities (P = 0.03). CONCLUSION: Polymerase chain reaction assay appears to be able to detect fungi in chronic rhinosinusitis. SIGNIFICANCE: Fungi may not be implicated in the pathogenesis of most chronic rhinosinusitis. EBM rating: B-3b.     

Cytomegalovirus infection of renal allografts. Detection by polymerase chain reaction.

Early laboratory diagnosis of acute cytomegalovirus infection in renal transplant recipients is desirable but often difficult. The polymerase chain reaction (PCR) technique for detecting CMV DNA, although it promises a high sensitivity, risks the possibility of detecting latent CMV infection and leading to false-positive results. To address this issue and the feasibility of applying PCR to renal biopsy specimens, we analyzed 37 renal allografts by PCR. Formalin-fixed or Bouin-fixed paraffin-embedded materials were employed, and primers from the LA (late-antigen) region of CMV were used. Amplified products were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot analysis. Of 21 nephrectomy samples, three showed CMV-specific amplified products by PCR, but CMV inclusion bodies were detected histologically in only one of the three. Of 16 renal biopsies, three specimens were positive by PCR, with rare viral inclusions histologically identified in only one. All PCR-positive patients had clinically significant CMV disease as evidenced by positive CMV culture and/or seroconversion. In contrast, all CMV-seropositive patients without active viral disease had PCR-negative allografts. We conclude that PCR positivity in the renal allograft strongly correlates with active CMV disease but not latent infection. For the diagnosis of active CMV disease in patients with a renal allograft, PCR provides a means that is more sensitive and objective than histologic examination, more specific than serology, and faster than viral culture.     

Epididymo-orchitis caused by intravesically instillated bacillus Calmette-Guérin: Genetically proven using a multiplex polymerase chain reaction method

The intravesical instillation of bacillus Calmette-Guérin (BCG) is a standard therapy for superficial bladder carcinoma. Tuberculosis-like inflammation in the genitourinary tract is a serious complication of BCG. It can occur after a long interval from the cessation of the intravesical BCG therapy. If inflammation occurs, it is necessary to test whether the BCG strain has caused it or another mycobacterium species has. However, there has never been a report that proves BCG causes the inflammation, because BCG is difficult to differentiate from other strains of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by conventional tests, including regular polymerase chain reaction (PCR). We first present a case of epididymo-orchitis, which developed 31 months after the cessation of BCG therapy, detected using a multiplex PCR method as having been caused by BCG. Our report illustrates the efficacy of this method to detect the responsible microbe that is thought to be transmitted from the instillated BCG strain.     

PCR检测尿结核杆菌的临床应用价值

评价聚合酶链反应（ＰＣＲ）诊断泌尿系结核的临床应用价值，采用ＰＣＲ检测经病理证实为泌尿系结核９例，并报告１７例尿ＰＣＲ阳性的可疑肾结核抗痨治疗前后的临床情况，结果：９例泌尿系结核者，ＰＣＲ检测阳性７例；１７例尿ＰＣＲ阳性的可凝肾结核者经抗痨治疗３个月后，有１４例症状完全消失，尿常规转为正常，认为ＰＣＲ是临床快速诊断泌尿系结核的有效方法。     

应用聚合酶链反应检测生殖道溶脲脲原体的研究

采用培养法和聚合酶链反应（ＰＣＲ）检测了溶脲脲原体（Ｕｕ）标准菌株纯培养物、模拟临床生殖道Ｕｕ感染样本和５２例不育症病人的精液样本。结果培养法阳性的样本ＰＣＲ亦为阳性，并比培养法高出２～３个稀释度。若以培养法作为标准，ＰＣＲ的敏感性为１００％，特异性为８８％，准确性为９０％，提示ＰＣＲ是一种特异、灵敏和快速检测Ｕｕ感染的诊断方法。     

Langerhans cell histiocytosis and human herpes virus 6 (HHV-6), an analysis by real-time polymerase chain reaction.

Patients with Langerhans cell histiocytosis (LCH) usually present to orthopedic surgeons because this disease most commonly affects bone. The pathogenesis of LCH is unknown, although roles for environmental, infectious, immunologic, and genetic causes have been postulated. More specifically, there is limited data suggesting that human herpes virus 6 (HHV-6) may be a potential etiologic agent. Frozen biopsy material was obtained from 13 patients with LCH and 20 patients without the disease. After ensuring histologic adequacy of the material, the tissue was tested for HHV-6 by qualitative and quantitative real-time TaqMan PCR. Four of 13 patients with LCH had evidence of HHV-6 DNA in their tissue while 7 of 20 control patients tested positive for HHV-6 genome. Viral loads are reported for the positive patients; no statistical difference was observed in the presence or quantity of HHV-6 DNA found in either population, suggesting that the prevalence of HHV-6 in the tissue of LCH patients is the same as that found in tissue from individuals without disease.     

Detection of Brucella abortus B19 strain DNA in seminal plasma by polymerase chain reaction in Brazil

A total of 27 seminal plasma samples from cattle‐breeding farms or semen centres located in Minas Gerais, Brazil, previously negative by serological and tested positive for Brucella spp. with primer specific for the amplification of the gene virb5 by polymerase chain reaction (PCR ) were analysed for the detection of Brucella abortus DNA by PCR . It was found that nine samples (33.33%) contained B. abortus B19 strain DNA , two (7.40%) contained B. abortus DNA and five (18.51%) contained both DNA . The larger number of samples with B. abortus B19 strain DNA would explained by the environmental contamination by vaccinated females with persistent excretion or some illegal vaccination process. It is first reported of male bovines detected with both DNA .     

Prosthetic infection: improvement of diagnostic procedures using 16S ribosomal deoxyribonucleic acid polymerase chain reaction

Purpose

Prosthetic infection is the worst complication in joint arthroplasty. The diagnostic procedure is time consuming and in many cases unrewarding. The aim of this investigation was to raise the sensitivity of the diagnostic procedure.

Methods

Altogether, 229 implants were removed from 229 patients. Complete data from 157 patients could be analysed. On explantation of the respective arthroplasty, tissue was removed, puncture fluid aspirated and biofilm scratched from the implant surface with a surgical knife. Specimens were investigated with conventional culture methods and with 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and sequencing.

Results

In 123 cases, no pathogen could be identified by routine culture methods. In three of these culture-negative cases, bacteria could be identified with 16S rDNA sequencing of the removed biofilm. In 34 cases, bacteria could be identified with culture methods. In two of these cases, sequencing detected additional pathogens.

Conclusions

The process of 16S ribosomal deoxyribonucleic acid polymerase chain reaction (rDNA PCR) and sequencing of biofilm removed from the explanted prosthesis is an important addition to conventional culture methods in prosthetic joint infection. Polymerase chain reaction detects additional pathogens and improves diagnostic sensitivity. The examination of tissue, puncture fluid and biofilm should be performed in cases of prosthesis loosening and explantation.     

Repopulation of vascularized bone allotransplants with recipient‐derived cells: Detection by laser capture microdissection and real‐time PCR

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (±2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real‐time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex‐determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77–97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex‐mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1514–1520, 2009     

荧光定量聚合酶链反应检测大肠癌外周血角蛋白19、20的联合表达

目的研究大肠癌患者外周血CK19、CK20mRNA的表达,并探讨其临床意义。方法应用荧光定量聚合酶链反应(PCR)技术,检测大肠癌患者术前外周血CK19、CK20mRNA,并将其与临床、病理各项指标进行统计学分析。结果45例大肠癌患者外周血中CK19mRNA检测阳性率31.11%(14/45);CK20mRNA阳性率15.56%(7/45);联合检测CK19、CK20mRNA阳性率37.78%(17/45),较单项检测有明显提高。统计结果显示联合检测CK19、CK20mRNA与临床分期(χ2=4.54,P<0.05)、病理类型(χ2=5.66,P<0.05)、外周血CEA值(χ2=9.94,P<0.01)相关,差异有显著性。结论CK19、CK20mRNA是检测大肠癌微转移的标志物,对有外周血CK19、CK20RNA升高的患者应给予有效的免疫治疗、化疗和手术,并加以密切随访,以阻止微转移向转移灶的转变。     

Clonality studies in giant cell tumor of bone.

Genetic studies including chromosome analysis, telomere reduction and telomere activity, DNA microsatellites and loss of heterozygosity (LOH) studies have been performed on giant cell tumor (GCT) of bone however whether this primary skeletal neoplasm represents a monoclonal or polyclonal proliferation is unknown. Utilizing a new assay to study the polymorphic human androgen receptor locus (HUMARA), the ratio of maternal inactive X-chromosome to the paternal inactive X (Lyon hypothesis) is determined via a methylation--specific polymerase chain reaction (PCR) technique to detect X-chromosome polymorphisms. Characterization of the genetic tumorigenesis of this unpredictable neoplasm may lend insight into its biological behavior and offer improvements in therapeutic intervention, as new information emerges regarding osteoclastic bone resorption. Seventeen female patients with giant cell tumor of bone had their DNA harvested and their X-chromosome inactivation pattern and polymorphisms determined and compared to control. A polyclonal proliferation pattern was identified in all informative samples studied.