Establishment of a recombined mannose-binding lectin protein-magnetic beads-enriched binding recombinant enzyme-assisted polymerase chain reaction assay for Candida in blood samples / 中华流行病学杂志

Objective: To establish a nested recombinant enzyme-assisted polymerase chain reaction (RAP) technique combined with recombined mannose-binding lectin protein (M1 protein)-magnetic beads enrichment for the detection of Candida albicans (C. albicans) and Candida tropicalis (C. tropicalis) in blood samples for the early diagnosis of candidemia albicans and candidiemia tropicalis. Methods: The primer probes for highly conserved regions of the internal transcribed spacerregions of C. albicans and C. tropicalis were deigned to establish RAP assays for the detections of C. albicans and C. tropicalis; The sensitivity and reproducibility of nucleic acid tests with gradient dilutions of standard strains and specificity of nucleic acid tests with common clinical pathogens causing bloodstream infection were condcuted. M1 protein-magnetic bead enriched plasma C. albicans and C. tropicalis were used for RAP and PCR in with simulated samples and the results were compared. Results: The sensitivity of the established dual RAP assay was 2.4-2.8 copies/reaction, with higher reproducibility and specificity. M1 protein-magnetic bead enrichment of pathogen combined with the dual RAP assay could complete the detections of C. albicans and C. tropicalis in plasma within 4 hours. Fie the pathogen samples at concentration <10 CFU/ml, the number of the samples tested by RAP was higher than that tested by PCR after enrichment. Conclusion: In this study, a dual RAP assay for the detections of C. albicans and C. tropicalis in blood sample was developed, which has the advantages of accuracy, rapidity, and less contaminants and has great potential for rapid detection of Candidemia.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection / 生物医学与环境科学（英文）

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (

A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus / 生物医学与环境科学（英文）

OBJECTIVE@#Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease.@*METHODS@#A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay.@*RESULTS@#The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay.@*CONCLUSION@#A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.

Next-generation Sequencing Study of Pathogens in Serum from Patients with Febrile Jaundice in Sierra Leone / 生物医学与环境科学（英文）

OBJECTIVE@#People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus (YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice.@*METHODS@#In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV-negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice.@*RESULTS@#The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus (89.58%), Epstein-Barr virus (55.21%), hepatitis C virus (34.38%), rhinovirus (28.13%), hepatitis A virus (20.83%), coxsackievirus (10.42%), Ebola virus (8.33%), hepatitis E virus (8.33%), lyssavirus (4.17%), leptospirosis (4.17%), chikungunya virus (2.08%), Crimean-Congo hemorrhagic fever virus (1.04%), and hepatitis B virus (1.04%).@*CONCLUSION@#The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.

Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus / 生物医学与环境科学（英文）

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RTRAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39.0 °C for 30 min. The analytical sensitivity of the RT-RAA assay was 10 plasmid copies and 1.6 pfu per reaction with real-time fluorescence, and 1,000 plasmid copies per reaction with the LFD. No crossreaction with other control viruses was observed. Compared with the RT-qPCR assay, the RT-RAA assay demonstrated 100% sensitivity and 100% specificity for WNV.

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.</p><p><b>METHODS</b>A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.</p><p><b>RESULTS</b>A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.</p><p><b>CONCLUSION</b>The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.</p>

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.</p><p><b>METHODS</b>Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.</p><p><b>RESULTS</b>NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.</p><p><b>CONCLUSION</b>The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.</p>

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.</p><p><b>METHODS</b>In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.</p><p><b>RESULTS</b>Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.</p><p><b>CONCLUSION</b>MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.</p>

Visual detection of murray valley encephalitis virus by reverse transcription loop-mediated isothermal amplification / 生物医学与环境科学（英文）

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of Murray valley encephalitis virus (MVEV) infection. The reaction was performed in one step in a single tube at 63 °C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was 100 copies per reaction based on 10-fold dilutions of in vitro transcribed RNA derived from a synthetic MVEV DNA template. No cross-reaction was observed with other encephalitis-associated viruses. The assay was further evaluated using spiked cerebrospinal fluid sample with pseudotype virus containing the NS5 gene of MVEV.

Establishment and primary application of a novel resequencing pathogen microarray-based assay for detecting pathogens in patients with unexplained diarrhea / 病毒学报

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2).</p><p><b>METHODS</b>A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated.</p><p><b>RESULTS</b>The detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL.</p><p><b>CONCLUSION</b>A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).</p>

Advance in loop-mediated isothermal amplification technique and its applications in point-of-care testing platforms / 病毒学报

Loop-mediated isothermal amplification (LAMP) is a novel in vitro nucleic acid amplification method conducted under isothermal conditions with the advantages of high specificity, sensitivity, rapidity and easy detection. Since it was established in 2000, it has been widely applied in various fields of analytical science including the diagnosis of a variety of pathogens, identification of embryo sex, detection of genetically modified organisms and cancer gene identification. Additionally, significant progress has been made in the optimization of the LAMP method, such as accelerated reactions, simplified sample processing, the realization of multiplex amplification, and the enhanced specificity of reaction and detection methods. LAMP technology also shows much potential to be adopted as part of point-of-care testing platforms by the micromation, automation and integration with other technologies such as Lab-on-a-Chip and digital nucleic acid amplification. This review summarizes the latest advances in the LAMP technique and its applications in developing point-of-care testing platforms.

Pathogenic spectrum of enteroviruses associated with hand, foot and mouth disease by a GeXP™-based multiplex reverse transcription-PCR assay in Jinan, China, 2009-2012 / 病毒学报

We investigated the pathogenic spectrum of enteroviruses associated with hand, foot and mouth disease (HFMD) in Jinan, China. A total of 274 specimens with a clinical diagnosis of HFMD in Jinan from 2009 to June 2012 were used. A GenomeLab™ (GeXP)-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was employed to simultaneously detect 15 serotypes of human enteroviruses: human enterovirus (EV)71; coxsackievirus A (CVA)16, 4, 5, 6, 9, and 10; CVB1, 3 and 5; echovirus (Echo) 6, 7, 11, 13 and 19. Results showed that all samples were enterovirus-positive, with the most common serotypes being EV71 (25.18%) and CVA16 (16.06%), followed by CVA10 (14.23%), CVA6 (7.30%), CVB1 (1.09%), Echo6 (0.73%), CVA9 (0.36%), CVB3 (0.36%) and co-infections (5.11%). CVA10 and CVA6 had the third and fourth highest prevalence of pathogens for HFMD, respec- tively. The most prevalent season for CVA10 was from April to August, with a peak in April; for CVA6 it was from April to August, with a peak in June. This is the first report of the pathogenic spectrum of en- teroviruses associated with HFMD in Jinan using the GeXP-based multiplex RT-PCR assay. These data will provide the scientific evidence for the prevention and control of epidemics, as well as therapy for HFMD patients.

The novel multiplex PCRs and their applications in respiratory viruses diagnosis / 病毒学报

Acute respiratory tract infections (ARTIs) are widely distributed among the population, mainly caused by respiratory viruses. ARTIs are responsible for significant morbidity and mortality among the elderly and infants or young children, causing a serious economic burden. The rapid and accurate identifi cation of a pathogen will provide a guideline for the clinical diagnosis and therapy. Multiplex polymerase chain reaction (PCR) technologies combine the rapidness and high sensitivity of PCR with high through put, thus achieving the capability of detecting multiple pathogens simultaneously. The commercial kits based on these multiplex PCR methods allow to detect more than twelve respiratory viruses simultaneous ly, reaching the comparable sensitivities and specificities to those of real-time PCR. The recent progress of novel multiplex PCR assays and their principles as well as applications in respiratory virus diagnosis were reviewed in this paper.

Establishment of method and modification of colorimetric judgment on HIV-1 virus detection by reverse transcription loop-mediated isothermal amplification / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods for on-site HIV-1 detection.</p><p><b>METHODS</b>As for the real-time fluorescent RT-LAMP, we firstly tested the specificity and sensitivity, then explored its quantitative determination, and finally applied the method to the detection of 35 HIV-1 positive samples. For colorimetric judgment, after choosing different ameliorates to modify Hydroxynaphthol blue (HNB), we tested their real effects on coloration, and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.</p><p><b>RESULTS</b>The real-time fluorescent RT-LAMP showed great specificity of HIV-1, and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction. On testing 35 HIV-1-positive samples, the method could reach 100 percent detection rate. However, for the quantitative determination, the quantitative relation was not observed regarding the HIV-1 RNA of below 10(3) copies per reaction. Three modified HNB dyes with clear color variation between the reaction tubes of the negative and the positive were got in the study, and their sensitivities equaled to the level of agarose gel electrophoresis. Similarly, 100% (35/35) detection rate was reached when the colorimetric RT-LAMP with the modified dyes was applied to detect 35 HIV-1-positive samples.</p><p><b>CONCLUSION</b>The established real-time fluorescence method and the modified color judgment of RT-LAMP could be helpful for truly achieving rapid, accurate, and sensitive on-site detection of HIV-1.</p>

The establishment of a new resequencing pathogen microarray-based assay for detecting unexplained respiratory tract infections / 病毒学报

Resequencing Pathogen Microarray (RPM) is a new pathogen detection and identification technology based on DNA microarray. In order to apply RPM in the detection of unexplained infection and as a result, to improve the emergency response capacity, a new RPM-based respiratory pathogens detection assay was developed to simultaneously detect 19 common respiratory viruses, 9 influenza A viruses (Flu A),11 human rhinoviruses(HRV), 28 enteroviruses and 18 rare respiratory viruses. The specificity of multiplex system was examined by confirmed positive specimens for 16 common respiratory virus. The sensi-tivity was evaluated by serial ten-fold dilutions of plasmids or in vitro-transcribed RNA. RPM could detect and differentiate 16 virus types/subtypes at 10 - 1 000 copies/reaction level. Nucleic acids of 8 throat swabs with unexplained respiratory tract infections were pooled and detected by the new assay. The RPM result was verified by common PCR followed by sequencing as well as PLEX-ID (Abbott). Except for a false-positive of PIV1, no difference among the three assays was found. These results indicate the assay based on the new RPM is a highly sensitive, high throughput test for the detection of respiratory virus infections, which is significant for the management of emergent and epidemic infectious disease.

Visual detection of HIV-1 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HIV-1.</p><p><b>METHODS</b>RT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The performance of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR(qRT-PCR).</p><p><b>RESULTS</b>The RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100%, respectively,in comparison with those of qRT-PCR.</p><p><b>CONCLUSION</b>RT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.</p>

Development of a GeXP based multiplex RT-PCR assay for simultaneous detection of eight arboviruses related to encephalitis / 病毒学报

Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 10(2) copies/microL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.

Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification / 病毒学报

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.

Different strength intermittent treadmill training of growth period rats and related bone metabolism of the hormone influence / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the influence of different strength intermittent treadmill training of growth period rats on the bone metabolism, so as to provide the training intensity of teenagers to set theory support.</p><p><b>METHODS</b>Select 70 male four weeks Wistar rats according to body weight randomly divided into seven groups (n = 10): the control group and the exercise group. According to the VO2max the exercise group was divided into 6 groups: 65%, 70%, 75%, 80%, 85% and 90% group. Nine weeks treadmill training, training six days a week, each group of training three times, each time not less than 10min, the interval was 30 min. The last movement after 24 h, took the femur and blood to measured the bone mineral density (BMD), bone mass (BMC) and alkaline phosphatase (AKP), resist tartaric acid acidic phosphatase (Str-ACP).</p><p><b>RESULTS</b>1. The femoral BMD (0.1393 +/- 0.0031), BMC (0.4525 +/- 0.0335) of 70% group were significantly higher than those in the control group (BMD: 0.1200 +/- 0.0095, BMC: 0.3238 +/- 0.0485) and the other sports group (65% BMD:0.1339 +/- 0.0062, BMC: 0.4058 +/- 0.0492, 75% BMD: 0.1296 +/- 0.0015, BMC: 0.3869 +/- 0.0254, 80% BMD: 0.1223 +/- 0.0082, BMC: 0.3454 +/- 0.0483, 85% BMD: 0.1250 +/- 0.0044, BMC: 0.3731 +/- 0.0381, 90% BMD: 0.1171 +/- 0.0047, BMC: 0.3051 +/- 0.0286) (P < 0.05), the femoral BMD, BMC of 90% group were lower than those of the control group, the other in the exercise group were higher than those in the control group; 2. Serum AKP in the exercise group were significantly higher than those in the control group, the group of 65% (41.015 +/- 2.114), 70% (46.035 +/- 2.611), 75% (43.834 +/- 3.155), and 80% (38.043 +/- 4.073) were very significantly higher than those in the control group (26.875 +/- 1.188) (P < 0.01); 70% group and 75% group were significantly higher than those in the 80% group , 85% group and 90% group, while 70% group serum AKP level were significantly higher than those in 65% group (P < 0.05), it showed that 70% of the VO2 max training intensity of osteoblasts was most active. The serum Str-ACP of exercise group were significantly higher than those in the control group, along with the increase of the training intensity, serum Str-ACP level was rising and the group of 80% (22.430 +/- 1.591), 85% (23.990 +/- 1.870), and 90% (28.009 +/- 1.839) serum of Str-ACP were significantly higher than those in the group of 65% (18.503 +/- 2.429), 70% (16.447 +/- 2.120) and 75%(17.769 +/- 1.642) ( P < 0.05), the group of 90% serum Str-ACP were significantly higher than those in the group of 80% and 85% (P < 0.05).</p><p><b>CONCLUSION</b>The training of 70% of the VO2max, moderate intensity intermittent running, make the growth period rat bone mass and bone mineral density to increase obviously.</p>