[Analysis of the methods and quality assurance of metagenomic next-generation sequencing to detect the microbial cfDNA from blood samples in China].

Objective: To investigate the methods and quality assurance of metagenomic next-generation sequencing (mNGS) to detect the microbial cfDNA (mcfDNA) from blood samples in different laboratories across China. Methods: In October 2020, questionnaires about detecting mcfDNA in blood samples with mNGS were distributed to 80 laboratories across the country. The questionnaire included four parts: pre-analysis, during analysis, post-analysis, and carrying out of performance validation for mNGS. (1) Pre-analysis: the requirements for samples quality, such as collection, storage, the transportation conditions of samples; (2) During analysis: the extraction workflows of mcfDNA, the quality requirements of the library, the application of the sequencing platforms and the bioinformatics analysis pipelines; (3) Post-analysis: the standard of interpretation results for mNGS; (4) Carrying out of performance validation: the minimum detection limit for various pathogens. All laboratories are required to fill in the questionnaire according to the actual situation. The feedback data were summarized and analyzed. Results: The 80 laboratories included 20 medical centers and 60 independent medical laboratories. There were 80.0% (64/80) of laboratories indicated that both plasma and serum samples were used to detect mcfDNA in blood, and the rest of the laboratories (16/80, 20.0%) only used plasma samples. The sequencing platforms used by mNGS laboratories involved in the survey included illumina (49), Beijing Genomics Institute (16), Ion Torrent (13) and Nanopore sequencing (2). There were 87.5% (70/80) of laboratories used the integrated analysis tools built by the third-party laboratories, and other laboratories (12.5%, 10/80) independently built the analysis platform by open-source software. The interpretation criteria of mNGS results varied between laboratories, among which the normalized number of pathogen-specific sequences, relative abundance, genome coverage rate, and the detection of the microorganism in the negative control were the main factors considered by laboratories. Most laboratories (76.3%, 61/80) had carried out the performance validation for the mcfDNA mNGS workflows. The limit of detection of the laboratories-developed mNGS workflows for Gram-positive bacteria, Gram-negative bacteria, fungi, parasites, and other pathogens were mainly distributed at 10-100 copies/ml, DNA virus was mainly distributed at 500-1 000 copies/ml. Conclusions: The mNGS workflows of various laboratories are very different. In order to ensure timely and accurate testing results, every laboratory needs to actively optimize the mNGS testing procedures, improve quality assurance measures, and carry out performance validation before mNGS is widely used in clinical settings.

LncRNA RP11-567G11.1 accelerates the proliferation and invasion of renal cell carcinoma through activating the Notch pathway.

OBJECTIVE: In recent years, the death number of renal cell carcinoma (RCC) has been enhanced annually. The crucial function of long non-coding RNA (lncRNA) in the occurrence and progression of cancer is of great significance. However, the specific role of lncRNAs in the pathogenesis and prognosis of RCC has not been fully elucidated. Therefore, the aim of this study was to uncover the role of lncRNA RP11-567G11.1 in regulating the progression of RCC. PATIENTS AND METHODS: Relative expression level of RP11-567G11.1 in RCC tissues and cells was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The influences of RP11-567G11.1 on proliferative and invasive abilities of RCC cells were assessed. Subsequently, regulatory effects of RP11-567G11.1 on the viability and apoptosis of DDP-induced RCC cells were examined. Furthermore, the mRNA and protein levels of Notch pathway-related genes Jagged1/HES5/HEY1 in RCC were detected by qRT-PCR and Western blot, respectively. RESULTS: RP11-567G11.1 expression was significantly up-regulated in RCC tissues and cells. Meanwhile, RP11-567G11.1 was highly expressed in RCC patients with advanced stage. Knockdown of RP11-567G11.1 significantly attenuated proliferative and invasive abilities of 786-O and 769-P cells. Silence of RP11-567G11.1 attenuated viability, while it induced apoptosis in DDP-induced RCC cells. In addition, knockdown of RP11-567G11.1 remarkably down-regulated both mRNA and protein levels of Jagged1, HES5, and HEY1 in RCC. CONCLUSIONS: RP11-567G11.1 accelerates the proliferative and invasive abilities of RCC through activating the Notch pathway. Our findings suggest that it may be a new therapeutic target for RCC.

Nanocalorimeter platform for in situ specific heat measurements and x-ray diffraction at low temperature.

Recent advances in electronics and nanofabrication have enabled membrane-based nanocalorimetry for measurements of the specific heat of microgram-sized samples. We have integrated a nanocalorimeter platform into a 4.5 T split-pair vertical-field magnet to allow for the simultaneous measurement of the specific heat and x-ray scattering in magnetic fields and at temperatures as low as 4 K. This multi-modal approach empowers researchers to directly correlate scattering experiments with insights from thermodynamic properties including structural, electronic, orbital, and magnetic phase transitions. The use of a nanocalorimeter sample platform enables numerous technical advantages: precise measurement and control of the sample temperature, quantification of beam heating effects, fast and precise positioning of the sample in the x-ray beam, and fast acquisition of x-ray scans over a wide temperature range without the need for time-consuming re-centering and re-alignment. Furthermore, on an YBa2Cu3O7-δ crystal and a copper foil, we demonstrate a novel approach to x-ray absorption spectroscopy by monitoring the change in sample temperature as a function of incident photon energy. Finally, we illustrate the new insights that can be gained from in situ structural and thermodynamic measurements by investigating the superheated state occurring at the first-order magneto-elastic phase transition of Fe2P, a material that is of interest for magnetocaloric applications.

[The effect of curcumin on the expression of miR-155 to apoptosis and invasion of extravillus trophoblast cells treated by lipopolysaccharide].

Objective: To investigate the effect of curcumin on the expression of miR-155, apoptosis and invasion of extravillus trophoblast cells treated by lipopolysaccharide (LPS). Methods: Human trophoblast cells (HTR-8/SVneo cells) were divided into 4 groups, the curcumin + LPS group (pre-treated by curcumin of 12.5, 25, 50 µmol/L, then LPS of 100 ng/ml), the LPS group (100 ng/ml), the recombinant adenovirus group (miR-155, multiplicity of infection100) and the control group. The miR-155 level in HTR-8/SVneo cells was measured by real-time PCR, and the expression of NF-κB was analyzed by luciferase gene expression. The apoptosis of HTR-8/SVneo cells was tested by cell death detection ELISA and the level of NF-κB in HTR-8/SVneo cells was measured by western blot. In addition, transwell was used to test the invasive ability of HTR-8/SVneo cells in all the groups. Results: (1) The intracellular expression of miR-155 in the LPS group was (2.13±0.22) times of the control group (P<0.01); and the expressions of miR-155 in 12.5, 25, 50 µmol/L curcumin+LPS groups were (0.37±0.08) , (0.68±0.14) , (0.49±0.09) times as the LPS group, with statistically significant difference (P<0.05). (2) The expreesion of NF-κB in the LPS group was (2.25±0.56) times of the control group. The expreesion of NF-κB in the 12.5, 25, 50 µmol/L curcumin+LPS groups were (0.80±0.07) , (0.74±0.05) , (0.49±0.19) times to the LPS group, with statistically significant difference(P<0.01). (3)The p65 protein in the LPS group was (1.50±0.22) , significantly higher than the control group (0.95±0.25, P<0.01) . In 12.5, 25, 50 µmol/L curcumin+LPS groups, the p65 protein were (0.31±0.07) , (0.75±0.14) , (0.49±0.08) . Compared with the LPS group, p65 was down-regulated by curcumin, with statistically significant difference (P<0.05). (4) In the cell death detection ELISA, the A value in the control group, the LPS group and the recombined adenovirus group were (0.89±0.17) , (2.02±0.35) and (1.67±0.48) , respectively, with statistically significant difference (P<0.05). In the curcumin+LPS groups, when the curcumin concentrations were 25 or 50 µmol/L, the A value were (0.74±0.05) , (0.49±0.09) , respectively, compared with the LPS group(set as 1.00), with statistically significant difference (P<0.05). When the curcumin concentration was 12.5 µmol/L, the A value was (0.80±0.07) , with no statistical significance (P>0.05). (5) The transmembrane cells were (47±8), (60±14) in the LPS group and the recombined adenovirus group, respectively. Compared with the control group (169±18), the differences were statistically significant (P<0.05). In the curcumin + LPS groups, the transmembrane cells were (143±10), (137±10) when the curcumin concentrations were 12.5, 25 µmol/L, significantly higher than the LPS group (P<0.05) . The transmembrane cells were (55±7) when the curcumin concentration was 50 µmol/L, with no statistical difference(P>0.05). Conclusion: The treatment of curcumin could downregulate the expression of NF-κB/miR-155, thus inhibit NF-κB signal pathway and the apoptosis of extravillus trophoblast cells, and protect their invasive ability.

Nanostructured Stealth Surfaces for Visible and Near-Infrared Light.

So far, all previous attempts to apply nanostructures for perfect transmission have not achieved maximum transmittance beyond 99.5% due to the limited regularity of the nanoscale surface geometry: too low for many high-end applications. Here we demonstrate a nanostructured stealth surface, with minimal reflectance (<0.02%) and maximal transmittance (>99.8%) for a wavelength range, covering visible and near-infrared. Compared to multilayer thin film coatings for near-infrared applications our antireflective surfaces operate within a much broader wavelength range, are mechanical stable to resist human touch or contamination, show a 44% higher laser-induced damage threshold, and are suitable for bended interfaces such as microlenses as well.

Torque-mixing magnetic resonance spectroscopy.

A universal, torque-mixing method for magnetic resonance spectroscopy is presented. In analogy to resonance detection by magnetic induction, the transverse component of a precessing dipole moment can be measured in sensitive broadband spectroscopy, here using a resonant mechanical torque sensor. Unlike induction, the torque amplitude allows equilibrium magnetic properties to be monitored simultaneously with the spin dynamics. Comprehensive electron spin resonance spectra of a single-crystal, mesoscopic yttrium iron garnet disk at room temperature reveal assisted switching between magnetization states and mode-dependent spin resonance interactions with nanoscale surface imperfections. The rich detail allows analysis of even complex three-dimensional spin textures. The flexibility of microelectromechanical and optomechanical devices combined with broad generality and capabilities of torque-mixing magnetic resonance spectroscopy offers great opportunities for development of integrated devices.

Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.

BMP-2 promotes chondrogenesis of rat adipose-derived stem cells by using a lentiviral system.

Osteoporosis poses a major public health threat in aging societies. Adipose-derived stem cells (ADSCs) are multipotent adult stem cells that have the ability to yield mesenchymal stem cells, and have the potential to undergo osteogenesis and bone regeneration. Bone morphogenetic proteins (BMPs) have been demonstrated to upregulate bone gene expression after mechanical injury and to improve bone injury repair. This study aimed to produce BMP-2 expression in ADSCs by using lentiviral vectors. Subcutaneous adipose tissue from 4-week-old male Sprague-Dawley rats was used. Oil red O staining was used to detect adipocyte formation from ADSCs. Induction of ADSC osteogenesis was confirmed with Alizarin red S staining. The recombinant lenti-hBMP-2/neo was constructed to infect ADSCs, BMP-2 expression was measured by immunoblotting analysis, and cellular alkaline phosphatase levels were examined. We found that >70% of ADSC cells could be induced to differentiate into osteocytes or adipocytes. Under osteogenic induction, ADSCs showed increased intracellular calcium deposition, the formation of calcium tubercles, and the disappearance of cellular structures in calcium tubercles. After infection of ADSCs by lenti-hBMP-2/neo, BMP-2 was expressed after doxycycline induction. We, thus, conclude that ADSCs maintain vigorous growth ex vivo and possess stem cell-like properties. When infected with lenti-hBMP-2/neo, ADSCs can be induced to promote BMP-2 expression.

Optical racetrack resonator transduction of nanomechanical cantilevers.

Optomechanical transduction has demonstrated its supremacy in probing nanomechanical displacements. In order to apply nano-optomechanical systems (NOMS) as force and mass sensors, knowledge about the transduction responsivity (i.e. the change in measured optical transmission with nanomechanical displacement) and its tradeoffs with system design is paramount. We compare the measured responsivities of NOMS devices with varying length, optomechanical coupling strength gom, and optical cavity properties. Cantilever beams 1.5 to 5 µm long are fabricated 70 to 160 nm from a racetrack resonator optical cavity and their thermomechanical (TM) noise signals are measured. We derive a generic expression for the transduction responsivity of the NOMS in terms of optical and mechanical system parameters such as finesse, optomechanical coupling constant, and interaction length. The form of the expression holds direct insight as to how these parameters affect the responsivity. With this expression, we obtain the optomechanical coupling constants using only measurements of the TM noise power spectra and optical cavity transmission slopes. All optical pump/probe operation is also demonstrated in our side-coupled cantilever-racetrack NOMS. Finally, to assess potential operation in a gas sensing environment, the TM noise signal of a device is measured at atmospheric pressure.

miR-155* mediates suppressive effect of PTEN 3'-untranslated region on AP-1/NF-κB pathway in HTR-8/SVneo cells.

Among miRNAs, miR-155 is a known regulator of immune system. Accumulating studies have revealed the connections between miR-155 and activator protein 1 (AP-1)/nuclear factor (NF)-κB. However, miR-155*, a miR-155 paralog, has so far been less studied. Here we demonstrated that miR-155*, induced by lipopolysaccharide (LPS) in an AP-1/NF-κB dependent manner, played a positive feedback role in AP-1/NF-κB pathway via targeting interleukin-1 receptor-associated kinase M (IRAKM) and NF-κB inhibitor interacting Ras-like 1 (NKIRAS1) in trophoblasts. Our study further proved that miR-155*-targeted PTEN 3'-untranslated region (3'UTR) increased IRAKM and NKIRAS1 expression by competing for miR-155* binding, thereby suppressing AP-1/NF-κB activation induced by LPS.

Co-transfection of adeno-associated virus-mediated human vascular endothelial growth factor165 and transforming growth factor-ß1 into annulus fibrosus cells of rabbit degenerative intervertebral discs.

Intervertebral disc degeneration is a common condition that may lead to low back pain and radiculopathy. Understanding the pathophysiology and cellular and molecular events of degenerative disc disease has resulted in the proposal of a gene therapy approach to halt and reverse disc degeneration. We explored the feasibility of reversing intervertebral disc degeneration using human vascular endothelial growth factor165 (hVEGF165) and transforming growth factor-ß1 (TGF-ß1) gene therapy. hVEGF165 complementary DNA was obtained from pcDNA3(+)-hVEGF165 and cloned into adeno-associated virus (AAV)-pSNAV plasmids to construct the recombinant plasmid, AAV-pSNAV-hVEGF165. After identification through restriction enzyme digestion and DNA sequencing, the AAV-pSNAV-hVEGF165 was transfected into HEK293 cells and vascular endothelial cells. Protein expression of hVEGF165 was detected using a fluorescent immunohistochemical assay, and the effect of hVEGF165 on vascular endothelial cell proliferation was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Packaged AAV-hVEGF165 and AAV-TGF-ß1 were co-transfected into the annulus fibrosus cells of degenerative intervertebral discs. hVEGF165 and TGF-ß1 expression by annulus fibrosus cells and the effect of the co-transfection on the level of collagen type I protein expression by annulus fibrosus cells were detected with Western blot. The results of restriction enzyme digestion and DNA sequencing confirmed that AAV-pSNAV-hVEGF165 plasmids were constructed. The fluorescent immunohistochemical results confirmed hVEGF165 protein expression. The MTT results showed that the hVEGF165 protein promoted vascular endothelial cell proliferation. Biologically active AAV-hVEGF165 and AAV-TGF-ß1 were successfully constructed. Western blot confirmed hVEGF165 and TGF-ß1 expression in annulus fibrosus cells and demonstrated that the level of collagen type I protein expression was significantly higher in annulus fibrosus cells co-transfected with both AAV-hVEGF165 and AAV-TGF-ß1 compared with that in cells transfected with AAV-hVEGF165 or AAV-TGF-ß1 alone. hVEGF165 has a synergistic effect with TGF-ß1 that promotes the expression of collagen type I protein in annulus fibrosus cells from degenerative intervertebral discs.

Near-infrared characterization of gallium nitride photonic-crystal waveguides and cavities.

We report the design and optical characterization of fully suspended wire waveguides and photonic crystal (PhC) membranes fabricated on a gallium nitride layer grown on silicon substrate operating at 1.5 µm. W1-type PhC waveguides are coupled with suspended wires and are investigated using a standard end-fire setup. The experimental and theoretical dispersion properties of the propagating modes in the wires and photonic-crystal waveguides are shown. Modified L3 cavities with quality factors of up to 2200 and heterostructure cavities with quality factors of up to 5400 are experimentally demonstrated.

MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1.

MiR-155 is known to participate in various cellular processes by targeting gene expression. We previously revealed a link between miR-155 and perturbation of trophoblast invasion and differentiation. This study aimed to investigate the target molecule(s) of miR-155 on the influence on the proliferation and migration of trophoblast cells. Bioinformatics analysis showed that, at the 3' untranslated region (UTR) of cyclin D1, six bases are complementary to the seed region of miR-155. Luciferase assays and cyclin D1 3'UTR transfection assays validated that cyclin D1 3'UTR was the target of miR-155 in HTR-8/SVneo cells. Overexpression of miR-155 in HTR-8/SVneo cells reduced the level of cyclin D1 protein, decreased cell proliferation and invasion, and increased cell number at the G1 stage. Furthermore, the increased expression of miR-155 also regulated the protein levels of kinase inhibitory protein p27 and phosphorylated cytoskeletal protein filamin A. In conclusion, we found that cyclin D1 may be a target of miR-155 in HTR-8/SVneo cells, and demonstrated a negative regulatory role of miR-155 involved in cyclin D1/p27 pathway in proliferation and migration of the cells.

NUR77 inhibits the expression of TIMP2 and increases the migration and invasion of HTR-8/SVneo cells induced by CYR61.

OBJECTIVE: To investigate the function and mechanism of CYR61 on the migration and invasion of the trophoblast cell line, HTR-8/SVneo cells. STUDY DESIGN: The mRNA and protein levels of NUR77 in the placentas of normal and preeclampsia (PE) women were evaluated using real-time PCR and Western blot, respectively. Paraffin-embedded tissues were processed for localization of NUR77 protein in placental villus by immunohistochemistry. HTR-8/SVneo cells were cultured in the presence of CYR61, Ad-NUR77 or a small interfering RNA for NUR77 (Ad-sinur77). The expression of NUR77 in the HTR-8/SVneo cells was detected and the effects of CYR61 on the migration and invasion of HTR-8/SVneo cells were assessed in wound-healing and transwell experiments, respectively. Gelatin zymography was used to measure the MMP2 release in HTR-8/SVneo cells. RESULTS: NUR77 is significantly decreased in the placenta of women with PE compared with the levels during a normal pregnancy. CYR61 can significantly increase the expression of NUR77 in HTR-8/SVneo cells. CYR61, as well as NUR77, can promote HTR-8/SVneo cells migration and invasion, which can be blocked by Ad-sinur77. Both CYR61 and Ad-nur77 reduced the mRNA expression of TIMP2 in HTR-8/SVneo cells. CONCLUSIONS: CYR61 may promote HTR-8/SVneo cells migration and invasion through the upregulation of NUR77, leading to the increase of MMP2 release and the downregulation of TIMP2 expression.

Treatment of angiostrongyliasis using a combination of albendazole and dexamethasone: the results of a retrospective and comparative study.

As the information available on the treatment of angiostrongyliasis with a combination of albendazole and dexamethasone is limited, the efficacy of such therapy was assessed using data collected during the 2006 outbreak of angiostrongyliasis in Beijing. In a retrospective and controlled study, 35 patients treated with albendazole-dexamethasone (given 20 mg albendazole/kg and 3 mg dexamethasone daily for 7 days) were compared with 34 controls who were treated only symptomatically (with acetaminophen or other drugs). Compared with the controls, the patients given the combination were less likely to have headaches after 7 days (P = 0·038), tended to have headaches that cleared quicker (P = 0·010), and received fewer doses of acetaminophen (P = 0·036). Since no serious adverse effects were observed, a 1-week treatment with a combination of albendazole and dexamethasone appears both safe and beneficial in the treatment of angiostrongyliasis.

[Expression, characterization of recombinant human soluble BAFF secreted from CHO cell].

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 microg/ml to 0.55 microg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.

Perpendicular spin torques in magnetic tunnel junctions.

We quantitatively determine a perpendicular spin torque in magnetic tunnel junctions by measuring the room-temperature critical switching current at various magnetic fields and current pulse widths. We find that the magnitude of the torque is proportional to the product of the current density and the bias voltage, and the direction of the torque reverses as the polarity of the voltage changes. By taking into account the energy-dependent inelastic scattering of tunnel electrons, we formulate the bias dependence of the perpendicular spin torque which is in qualitative agreement with the experimental results.

Differential inactivation of Listeria monocytogenes by D- and L-lactic acid.

AIMS: To determine inactivation of Listeria monocytogenes by the two lactic acid isomers. METHODS AND RESULTS: The survival of four strains with varying sensitivity to acid was determined following treatment with L- or D-lactic acid at 100 mmol l(-1) (pH 3.7) or HCl at pH 3.37. There was some, but not complete, similarity in the relative sensitivity of the four strains to the two types of acid. All strains were most sensitive to D-lactic acid, which gave 0.6-2.2 log units greater reduction than L-lactic acid midway in the inactivation curves. Even very low concentrations of the two isomers had an immediate effect on pH(i) which was identical for the two isomers. CONCLUSIONS: The results show that L. monocytogenes is more sensitive to D- than to L-lactic acid; however, this difference is less than the strain variation in L-lactic acid sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work has implications for the application of lactic acid for food preservation as well as for the understanding of the antibacterial mechanisms of weak organic acids.

[Construction of a novel Bm NPV Bac to Bac system].

A Bi-Shuttle vector Bm-Bacmid was constructed by co-infecting Bm N cells with wild type genomic DNA from BmNPV and Ac-Bacmid DNA. It could not only replicate in E. coli cells as a large plasmid and but also remain infectious when induced into Bm N or Sf9 cells. Recombinant virus rBmHBe was obtained after transposition of a donor plasmid carrying Hepatitis Be antigen gene (HBeAg) into att Tn7, and was demonstrated by Southern blotting. SDS-PAGE analysis showed that HBeAg gene were highly expressed in Bm N cells. By ELISA testing, the highest antigenecity titer of HBeAg protein in cell cultural medium was up to a dilution of 1:32,000. Although HBeAg protein also presented in the Bm N cells the titer was only 1:2000. The HBcAg protein was much fewer than HBeAg (< 1:160) whatever in culture medium and in cells. The results showed that Bm N cells was able to recognize the signal peptide sequence and cut it correctly for HBeAg protein's excreting production.

[Detection of Plasmodium falciparum using the plasmid Pf rep 20 probe (preliminary report)].

A cloned repetitive DNA sequence (rep 20) labeled with 32P was evaluated as diagnostic probe for P. falciparum in 39 blood samples from Hainan and Yunan provinces. Its specificity and sensitivity were studied and compared with total genomic DNA probe isolated from P. f. Hainan Fcc1/HN isolate. The pPF rep 20 probe recognized the DNA of P. f. Hainan, Yunnan and Anhui isolates, but did not hybridize with the DNA of P. vivax, P. cynomolgi, P. knowlesi and P. yoelii. The genomic probe hybridized with the DNA of 3 isolates and cross-hybridized with the DNA of all the other Plasmodium species tested, but did not hybridize with host DNA. The plasmid rep 20 probe was able to detect parasitemia level of 0.001% in 20 microliters blood from culture and 10pg DNA of 3 P. f. isolates after 3 days film exposure. It could hybridize with the blood samples of P. f. patients from Hainan and Yunnan with a sensitivity of 95% (37/39). The genomic probe could detect the same parasitemia and DNA levels as plasmid rep 20 probe for Yunnan and Anhui isolates, and 0.0001% parasitemia and 1 pg DNA for Hainan isolate. It had a sensitivity of 97% (38/39) when used to detect patient samples. The results indicated that plasmid rep 20 probe was specific and rather sensitive to DNA of P. falciparum isolates from China and may be useful in epidemiological studies.