Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System / 华中科技大学学报（医学）（英德文版）

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance.Therefore,the development of methods for mutation detection characterized with straightforward,highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed.Although some of the currently available methods have shown very encouraging results,their discrimination efficiency is still very low.Herein,we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination,which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1％ within 20 min.The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15-38.48.The method is sequence independent,which assures a wide range of application.The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients’ acute phase serum & CSF samples.

Background & objectives: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. Methods: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. Results: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O’Nyong Nyong, Semlinki forest and Sindbis viruses. Interpretation & conclusion: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Detection of alphaB-crystallin mRNA using Single-stranded DNA Probe in Oligodendrocytes of the Developing Chick Retina / 체질인류학회지

In situ hybridization (ISH) using single-stranded DNA probe (ssDNA probe) is a useful method for observing the specific transcripts in cells, since it is convenient to prepare probe which is specific and sensitive. In this study, ssDNA probe for detection of alphaB-crystallin (aBC) mRNA, transcript of a heat shock protein, was prepared and aBC mRNA-expressed cells were spatiotemporally observed in the retina of the developing chick embryos. Single-stranded antisense probe produced by reverse transcription and polymerase chain reaction was identified as a specific probe for aBC mRNA in comparison to negative control using sense probe and immunohistochemistry for aBC protein. In the ISH experiment, aBC mRNA was expressed only in the peripapillary glial cells which are a specific cell type located in the avian retina adjacent to the optic nerve at E12 and E14 retinas. At E16, a small number of aBC mRNA-expressed cells were identified in the nerve fiber layer (NFL) of the retina. At E18, aBC mRNA-expressed cells were observed in the ganglion cell layer (GCL) as well as the NFL. At E20, the number of aBC mRNA-expressed cells was increased in the GCL and the NFL. Based on the same localization of nkx2.2 immunoreactive cells and aBC mRNA-expressed cells, aBC mRNA-expressed cells were identified as oligodendrocytes. These results indicate that ssDNA probe for aBC mRNA detection is very useful tool for oligodendrocyte research such as distribution, migration and differentiation of the cells.

Exactitud del test ADN-HPV para la detección de la enfermedad cervical de alto grado (NIC 2+) en mujeres con anormalidades citológicas (ASC-US y LSIL), afiliadas a la seguridad social en Bogotá (Colombia) / Accuracy of HPV-DNA test for detecting high-grade disease (CIN 2+)in women with minor cytological abnormalities (ASC-US and LSIL)

Objetivo: evaluar la exactitud de la prueba ADNHPV en el diagnóstico de lesiones premalignas cervicales de alto grado (HSIL, por sus siglas en inglés) en mujeres con alteraciones citológicas ASC-US (células escamosas atípicas de significado indeterminado) y LSIL (lesión escamosa intraepitelial de bajo grado). Metodología: estudio de validez diagnóstica y de cohorte transversal, realizado en mujeres con diagnóstico citológico de ASC-US o LSIL entre octubre de 2006 y enero de 2008, pertenecientes al Programa de Tamizaje de Cáncer de Cérvix de una aseguradora privada en Bogotá (Colombia). Se comparó el resultado de la colposcopia y de la prueba de ADN-HPV con la patología del cérvix como patrón de oro. Resultados: de las 429 mujeres, 344 (80,2%) presentaron ASC-US y 85 (19,8%) LSIL. Las prevalencias de infección por HPV de alto riesgo fueron 52,9% y 75,7% en pacientes con reporte citológico de ASC-US y LSIL, respectivamente. A las 379 de las 425 pacientes se les practicó biopsia, 24 (6,3%) dieron positivas para HSIL. La sensibilidad de la prueba ADN-HPV para detectar lesiones de alto grado (NIC 2+) en las mujeres con reportes de citología ASC-US y LSIL fue 88% y la especificidad fue 44%. En 21 (87.5%) de los 24 casos de HSIL se detectó la presencia de HPV de alto riesgo. Conclusión: debido a que la prueba ADN-HPV tiene una sensibilidad superior a la citología cervicovaginal (CCV), ésta puede considerarse una alternativa útil para estratificar el riesgo y mejorar la aproximación diagnóstica de lesiones premalignas del cuello uterino en mujeres con reporte de ASC-US.

Objective: evaluating the accuracy of the HPV DNA test as a complementary test for diagnosing highgrade cervical disease (high-grade squamous intra epithelia lesions-HSIL) in women with minor cytological abnormalities (atypical squamous cells of undetermined significance ASC-US) and low-grade squamous intraepithelial lesions (LSIL). Methodology: a diagnostic validity study based on a cross-sectional design was applied to 429 women who had had a cytological report of ASC-US and/or LSIL who were attending a health maintenance organisation’s cervical cancer screening programme in Bogotá, Colombia between January 2006 and October 2008. Colposcopy reports and HPV-DNA testresultswerecomparedwithpathologicalreports which were considered the gold standard. Results: 344 (80.2%) of the 429 women had a cytological report of ASC-US and 85 (19.8%) of them one for LSIL. High-risk HPV infection prevalence was 52.9% and 75.7% in patients having an ASC-US and LSIL report, respectively. A biopsy specimen was obtained in 379 of the 429 participants and 24 high-grade cases (6.3%) were diagnosed. DNA-HPV test sensitivity was 88% and specificity was 44% for detecting high-grade disease (CIN 2+) in women having an ASC-US and LSIL cytology report . The presence of high-risk HPV virus was detected in 21 of the 24 HSIL cases (87.5%). Conclusion: the DNA-HPV test’s higher sensitivity compared to the PAP smear (due to high NPV) means that it could be considered a useful tool for stratifying risk and improving the diagnostic approach to premalignant lesions of the uterine cervix in patients having a cytological report of ASC-US.

Mechanism of recombinant human bone morphogenetic protein-2 in repairing hematopoietic injury in mice exposed to γ-rays / 中华放射医学与防护杂志

Objective To investigate the mechanism of recombinant human bone morphogenetic protein-2(rhBMP-2)in repairing hematopoietic injury in mice irradiated with γ-ray.To prepare SRY gene probe and study the effect of rhBMP-2 in repairing hematopoietic injury in mice by in situ hybridization.Methods Twenty-two BALB/c female mice were randomly divided into the irradiated group and BMP treated group,respectively.Bone marrow cells of normal male mice were transplanted into 22 female mice post-irradiation to 8.5 Gy of 60 Co γ rays.The left femurs of the survived female mice were re-irradiated with 9 Gy 14 days later.Mice in BMP treated group were given rhBMP-2 20 mg/kg while those in control group were treated with 0.9% saline by intraperitoneal injection every day for 6 days.These mice were killed 14 days later and paraffin sections of femurs were made.The SRY gene was detected with in situ hybridization.Results There were more positive blots in the left femurs of the mice in irradiated group than those in BMP treated group(T=155.0,P＜0.05).The number of positive blots between the left femurs of the mice in irradiated group and the right femurs of the mice in two groups was not significantly different(T=92.0,78.5,P＞0.05).The number of positive blots in the left femurs of the mice in BMPtreated group was significantly less than those in the right femurs of the mice in two groups(T=155.0,55.0,P＜0.05).Conclusions No donor cell of male mice was detected in the left femurs of BMP treated group,suggesting that rhBMP-2 promoted the restoration of residuary bone marrow cells.Thus,rhBMP-2 promotes the proliferation or differentiation of residuary mesenchymal stem cells,improves hematopoietic microenvironment and accelerates the hematopoietic restoration.

Utility of RT-PCR-based Dot-blot Hybridization for Detecting and Genotyping Echoviruses

We attempted to detect and identify virus types quickly by improving an RT-PCR-based dot-blot hybridization test for echoviruses, important human pathogens mainly causing aseptic meningitis. This test was applied to reference viruses of seven echovirus serotypes prevalent in Korea (E6, 7, 9, 11, 13, 25, and 30) and seventy isolates of echovirus isolated in Korea between 2002 and 2004. The primers for target DNA and hybridization probes (25mer, 50mer, and 70mer) were designed within the VP1 region of the echovirus. In RT-PCR, a nonradioactive digoxigenin-DNA labeling mix was added instead of dNTP to initiate PCR. The PCR product was then hybridized against 25mer, 50mer, and 70mer probe DNA spotted on nylon membranes and the reaction was observed. To investigate the optimal conditions for hybridization, various concentrations of target DNA (0.1, 1, 10, and 100 ng/micron l), size of probe DNA (25mer, 50mer, and 70mer), concentrations of probe DNA (10~50 pM), and reaction time were included. In the test zone, the optimal condition in terms of time and cost was a reaction time of 1 h with 10 ng/micron l target DNA concentration and 10 pM of a 50mer probe. We found 100% diagnosis of the serotypes for seven reference echoviruses and 90% (63/70) sensitivity for clinical isolates. Also, tests with this probe for reactivity with seven reference echoviruses by using DNA chips showed that diagnostic identification was possible without other serotype cross-reactivity. Therefore, efficiency analysis of probe and target DNA on clinical specimens by using dot-blot analysis indicated that this system can be applied to the prestages of the DNA chip and that the dot blot analysis itself can be used in applications to develop a tool for diagnosing specific viral serotypes.

Rapid detection of methicillin-resistant staphylococci by DNA probe / 临床检验杂志

Objective To establish a dot blot hybridization technique for rapid detection of staphylococci and methicillin-resistant staphylococci.Methods Three pairs of primers were designed according to nuc gene of staphylococcus aureus,mecA gene of methicillin-resistance,tuf gene of staphylococci.Specific DNA probes were synthesized by polymerase chain reaction and labeled with biotin.The bacterial DNA inoculated on nitrocellulose filter was hybridized with these probes.The sensitivity and specificity were detected.Results The DNA probes with 270bp,310bp and 370bp were amplified by the three pairs of primers respectively.The probes were specific.Among 50 clinical isolates of staphylococcus aureus tuf and nuc gene were all positive and mecA gene in 22 isolates were positive.Positive rate of tuf,nuc and mecA gene in 30 staphylococcus epidermidis were 100%,0 and 30% (9/30) respectively.No hybridization in other non-staphylococci occurred.The established method could detect as low as 1ng of bacterial DNA.Conclusion The dot blot hybridization is of high value in rapid,effective identification of methicillin-resistant staphylococci.

Isolation of Nontuberculous Mycobacteria by DNA Probe and Clinical Characteristics of Patients with NTM Pulmonary Disease / 결핵및호흡기질환

BACKGROUND: Nontuberculous mycobacterial (NTM) infections are increasingly being recognized as a cause of chronic pulmonary disease. This study describes the prevalence of NTM species from clinical specimens and the clinical characteristics of NTM pulmonary disease. MATERIAL AND METHODS: The NTM isolated from March 2003 to December 2003 at the Kosin Medical Center were identified using an oligonucleotide chip containing the internal transcribed space (ITS) sequence. The medical records of the patients with the NTM isolates, who fulfilled the 1997 ATS diagnostic criteria for NTM pulmonary disease, were analyzed, retrospectively. RESULTS: Twenty four species (24.2%) of NTM were isolated from 99 cultured AFB specimens. M. avium complex (MAC) (13 isolates), M. szulgai (3), M. kansasii (2), M. malmoense (2), M. abscessus (1), M. chelonae (1), M. scrofulaceum (1), and unclassified (1). Of the 23 patients with isolated NTM, 11 patients were found to be compatible with a NTM pulmonary infection according to the ATS criteria; MAC was found in 6 cases (54.5%), M. szulgai in 2 cases (18.2%), and M. abscessus, M. szulgai, M. kansasii and M. malmoense in 1 case each (9.1%). Ten patients (91%) were male and the median age at diagnosis was 61 years. In the pre-existing diseases, malignant disease was found in 6 cases including 5 patients with lung cancer, and history of old pulmonary tuberculosis was identified in 4 cases. The radiological patterns showed lung destruction lung in 3 cases, a cavitary mass in 3 cases, a nodular pattern in 2 cases, and reticulonodular, consolidation and a bronchiectasis pattern were in 1 case each. CONCLUSION: Various types of NTM pulmonary diseases were`found in a tertiary hospital at Busan, Korea. The NTM pulmonary diseases were caused by MAC, M. szugai, M. kansasii, M. malmoense, M. abscessus, M. chelonae, and M. scrofulaceum in the order of frequency.

Study on isolation of Prevotella nigrescens 9336-specific DNA probes using random cloning method / 대한치주과학회지

The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.

Study on development of DNA probe for identification of Prevotella intermedia G8-9K-3 / 대한치주과학회지

The purpose of this study is to develop species-specific DNA probe for detection and identification of Prevotella intermedia (P. intermedia) G8-9K-3. This study procedure includes (1) whole-genomic DNA extraction of P. intermedia G8-9K-3 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot hybridization, (4) confirmation of strain-specific DNA probe by Southern blot hybridization, (5) determination of nucleotide sequences of strain-specific DNA probe. Twenty-eight recombinant plasmids containing Hind III-digested DNA fragments of P. intermedia G8-9K-3 were obtained. Reverse dot Hybridization and Southern blot analysis data showed that one of them, Pig3, could be P. intermedia G8-9K-3-specific DNA probe. This datum indicates that this Pig3 DNA probe could be useful in detection and identification of the P. intermedia G8-9K-3 strain.

Detection of Shiga Toxin-Producing Escherichia coli by in Stitu hybridization and sequence Analysis of Stx2 / 대한임상미생물학회지

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.

Detection of Shiga Toxin-Producing Escherichia coli by in Stitu hybridization and sequence Analysis of Stx2 / 대한임상미생물학회지

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.

Screening of Specific Probes from the DNA Library of Cryptococcus neoformans / 中华皮肤科杂志

To screen specific DNA probes by double hybridization from the constructed DNA li- brary of serotype A Cryptococcus neoformans.On the basis of the nucleotide sequence of vector pUC18, a pair of primers was synthesized.The insert fragments were amplified from the library on a PCR pro- cessor.The PCR products were spotted onto hybond-N~+ membranes.All inserts amplified from the ge- nomic library by PCR were screened by dot blot with 26 ~(32)P-labelled DNA from infectious agents for dif- ferentiation and from healthy persons.Twenty-eight candidate clones were obtained.The twenty-eight clone inserts got from low melting point agar were further characterized by dot blot with above 27 kinds of DNA for differentiation.Three specific DNA probes from the library of serotype A Cryptococcus neo- formans were obtained.Colony pCNIIA6 was serotype A-specific,which gave signals only with sertype A strain and did not cross hybridize with other DNAs.Colony pCNIIB5 was species-specific which gave signals only with DNA from Cryptococcus neoformans.Colony pCNIIIG1 was specific for var.neofor- mans,which gave signals only with serotype A and D strains.We can make a rapid diagnosis of Crypto- coccus neoformans infection at an early stage and distinguish the variants of C.neoformans and C.gattii using above specific probes.

STUDY ON HERPES SIMPLEX VIRUS TYPING WITH MOLECULAR BIOLOGICAL METHODS / 微生物学通报

A total of twenty eight HSV isolates from the patients were typed by using HSV typespecific monoclonal antibodies (McAb). Comparison of typing results with McAb in three Sero-lminunological methods with molecular hybridization indicated 100% concordance in the results. But the typing rates were quite different among the various methods (ELISA 64%, IFA and EIA 82%, Hybridization 100%). The results demonstrate that the molecular hybrydization with HSV type-specific probes was highly sensitive and specific, and the method of EIA with HSV type-specific McAb was accurate, cheap, rapid and practical.

COLONY HYBRIDIZATION WITH BIOTIN LABELED DNA PROBE / 微生物学通报

A simple, rapid method for colony hybridization has been developed. The DNA probes were labeled by digo xigenin. The signal of hybridization was detected by streptavidin and poly (AP) system. The results showed that this method is sensitive, specific and repoducible, it can be used for colony hybridization instead of isotopic.

APPLIED STUDY ON SINGLE LOCUS DNA PROBE FOR EARLY PREGNANCY PATERNITY TEST / 中国法医学杂志

30 chorinic villi in early pregnancy (8-10 weeks)and their parent blood samples were analysedusing the method of restriction fragment length polymorphisms with single locus DNA probes of PAC255 and PAC256. Based on the gene frequencies of these two loci in Chinese and the relativechance of obligatory gene in each triplet, the cumulative parternity index and cumulative relativechance of paternity were calculated. The range of the former is 56. 80- 8333. 50, and the later is 98.27 - 99. 99 %. According to the international standard of paternity test, in the 30 pedigrees, 20 cases(66. 7 % )were assured for their paternity. It is showed that the combination of PAC255 and PAC256single locus DNA probes for the paternity test of early pregnancy is very available.

FURTHER REPORT ON APPLICATION OF DNA PROBE IN DIAGNOSIS OF VIVAX MALARIA INFECTION / 中国寄生虫学与寄生虫病杂志

Southern blotting with a labeled and linearized pUC 19 DNA containing a specific fragment of 0. 24 kb DNA of Plasmodium vivax asexual blood stages (kindly offered by Dr. C. Kidson )was used for further identification of blood samples showing positive reaction by dot-blot hybridization. The results showed that those with positive reaction from patients with P. vivax, with P. falciparum or with fever but with negative microscopic findings were also positive by Southern blotting. It was confirmed that some of those positive with P. falciparum were likely to infect P. vivax at the same time. So did a part of those with fever but negative in the blood films (Figs. 1,2).

PATERNITY TEST STUDY BY DNA FINGERPRINTS AND SEROLOGICAL TESTING / 中国法医学杂志

Comparison between the DNA fingerprinting and the serological method used for paternity test is presented. 32 cases of disputed paternity were tested using DNA fingererints with MYO minisatellite DNA probe and blood typing. The results of DNA fingerprinting test correlated with those of blood typing. 8 alleged fathers were excluded while 24 were confirmed. The relationship between the results from two methods was discussed.