General hapten skeleton motivated duplex-immunoassay for emergent bisoxatin adulterants in slimming foods.

Adulterating hazardous bisoxatin (BSO) and bisoxatin acetate (BSOA) in slimming foods poses a threat to public health. A rapid synchronous detection method is urgently needed. Herein, the precise design of four novel haptens based on the general skeleton of BSO and BSOA was driven by computer-chemical visualization strategy, which was used to raise monoclonal antibody (mAb) toward both target compounds. The generated mAb 1F1 recognized BSO and BSOA with maximal half-inhibitory concentration (IC50) of 0.26 and 16.85 ng/mL, respectively. The molecular mechanism governing the duplex-recognition of mAb was elucidated by homology modeling and molecular docking. Finally, an immunochromatography (ICA) was developed for identifying BSO and BSOA, demonstrating a detection capability for screening (CCß) estimated to be 10-500 ng/g in candy tablets, jellies, and oral liquids. This study provides a robust approach for determining adulteration in food and offers insights into hapten design to improve antibody recognition spectrum.

Authenticating Emergent Adulterant 6-Benzylaminopurine in Bean Sprouts: Virtual Hapten Similarity Enhanced Immunoassay.

6-Benzylaminopurine (6-BA), a plant growth regulator with cytokinin-like properties, was recently reported to be illegally used in bean sprouts to increase their commercial appearance. It is still nevertheless challenging to quickly detect this adulteration. In this work, four novel haptens (haptens 1-4) of 6-BA were rationally designed with computer-assisted modeling analysis and then synthesized for use as immunizing haptens to produce antibodies. One of two obtained antibodies showed high sensitivity and specificity toward 6-BA. Based on the most sensitive anti-6-BA antibody, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was performed, which demonstrated a 50% inhibition concentration (IC50) of 1.18 µg/L and a limit of detection of 0.075 µg/L. The average recoveries of this icELISA for 6-BA of spiked samples ranged from 87.2 to 95.0% with a coefficient of variation of less than 8.7%. Furthermore, the blind samples were detected simultaneously by the method and HPLC-MS/MS, and the results showed good agreement with each other. Therefore, the proposed icELISA can facilitate the rapid surveillance screening of adulterated 6-BA in sprout vegetables.

Identifying fungicide difenoconazole as illegal growth regulator in vegetable: Computer-aided hapten similarity to enhance immunoassay sensitivity.

Difenoconazole, a fungicide with broad-spectrum properties, has recently been found to have been used illegally used as a plant growth regulator in Brassica campestris, with the intent of inducing thick stems and dark green leaves. However, analysts have encountered challenges in implementing a rapid surveillance screening approach for this purpose. In this study, a novel hapten was designed to improve the analytical performance of difenoconazole immunoassay. Specifically, the triazole of the original hapten was replaced with a benzene ring, guided by molecular simulation. This led to the development of a very sensitive antibody and the subsequent development of a competitive indirect enzyme linked immunosorbent assay (ciELISA) for the detection of difenoconazole in vegetable samples. The assay exhibited a working range of 0.16 ng mL-1 to 9.64 ng mL-1, with a detection limit of 0.05 ng mL-1. Upon analysis of blind samples, a strong correlation was observed between the ciELISA and HPLC-MS/MS methods. As a result, the proposed technique may prove to be an excellent tool for the rapid detection of difenoconazole overuse and adulteration in vegetables.

Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System.

BACKGROUND: Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. METHODS: In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. RESULTS: The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. CONCLUSION: Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

The establishment and clinical evaluation of a novel, rapid, no-wash one-step immunoassay for the detection of dengue virus non-structural protein 1.

Dengue fever is a highly endemic arthropod-borne viral disease in the tropical and sub-tropical countries and is rapidly becoming a global threaten. Diagnosis has been conducted by either traditional serological methods or molecular biological techniques. However, these methods are either labor-intensive, time-consuming or with multiple steps, which are not suitable for high throughput detection of large quantity of samples. In the current study, a novel, rapid, no-wash one-step amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) was developed and optimized for the diagnosis of dengue fever through the detection of dengue virus non-structural protein 1 (NS1). The linear range of the assay was determined to be 60,000 pg/mL to 200 pg/mL, with a lower detection limit of 127.45 pg/mL for NS1 protein. The precision of the assay was 8.24 % and 4.93 % for the high and low concentration. Clinical evaluation indicated that the sensitivity and specificity of the assay was 91.49 % and 81.54 %, respectively. This novel, rapid, no-wash one-step AlphaLISA assay is convenient and sensitive, which could be a good alternative for the screening of dengue fever in a high throughput format.

High HIV-1 prevalence and viral diversity among entry-exit populations at frontier ports of China, 2012-2016: A cross-sectional molecular epidemiology study.

The cross border movement of populations would potentially increase the transmission and spread of various diseases including HIV/AIDS, which needed strict surveillance. In the current study, a total of 2,961,530 specimens were collected between 2012 and 2016 from the entry-exit populations at frontier ports of China for epidemiology and molecular epidemiology study. Results showed that HIV-1 prevalence rate among these populations was significantly higher than that of other general populations in China (p < .001). Epidemiological investigation indicated that most of the HIV-1 infected participants were male and young general population, in contrast to intravenous drug users as revealed by previous studies. There were significantly more female, Chinese, and migrant labors in southeastern ports than in northwestern ports (p < .05). Molecular epidemiology study revealed that three subtypes C/BC, CRF01_AE and B dominated, with the emerging of novel unique recombinant forms and drug-resistant viruses among this population. Overall, the results suggests and calls for the shift of HIV-1 prevention projects' focus and the continuous monitoring of HIV-1 molecular epidemiology among entry-exit populations. These findings are useful for the generation of evidence-based biomedical and behavioral prevention programs to HIV/AIDS.

Near Full-Length Genomic Characterization of a Novel HIV-1 Unique Recombinant (CRF55_01B/CRF07_BC) from a Malaysian Immigrant Worker in Zhejiang, China.

Recombinant forms contribute substantially to the genetic diversity of human immunodeficiency virus type 1 (HIV-1). Here we report a novel HIV-1 recombinant detected from a comprehensive HIV-1 molecular epidemiologic study among cross-border populations in China. Near full-length genome (NFLG) phylogenetic analysis showed that the novel HIV-1 recombinant ZJCIQ15005, which was isolated from a Malaysian immigrant worker in Zhejiang, China, clustered with CRF55_01B reference sequences but set up a distinct branch. Recombinant analysis showed that the NFLG of ZJCIQ15005 composed of CRF55_01B (as the backbone) and CRF07_BC, with 12 recombinant break points observed in the pol, vif, vpr, tat, rev, env, nef, and 3'LTR regions. This is the first detection of a novel HIV-1 recombinant (CRF55_01B/CRF07_BC) in immigrant workers in China. The emergence of this recombinant may increase the complexity of the HIV-1 epidemic in China and suggests the importance of continuous surveillance of the dynamic changes of HIV-1.

Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens.

Complete nucleotide sequence analysis of the norovirus GII.17: A newly emerging and dominant variant in China, 2015.

Norovirus is an important pathogen which accounts for majority of the viral related acute gastroenteritis. Recently, a variant of genotype GII.17 was reported to be predominant over GII.4 and accounted for several acute gastroenteritis outbreaks in Asia. In the current study, the full genome of a norovirus strain ZHITHC-12 isolated during this outbreak period in China was identified and characterized. The viral genome was 7557 nucleotides in length and a phylogenetic analysis based on full length genome sequences indicated that ZHITHC-12 belonged to GII.17 genotype. A further phylogenetic analysis based on all available polymerase and capsid sequences showed that ZHITHC-12 was in Cluster III on both phylogenetic trees and grouped with other strains also isolated during 2013 to 2015. Moreover, homology modeling analysis based on GII norovirus capsid 5BSX template revealed that substitutions, mutations, and more importantly, deletions and insertions, occurred at or near the putative epitopes and histo-blood group antigen (HBGA) binding sites in its protruding P2 domain, which might confer new antigenic or biological properties for this novel variant. In summary, the first full genome and capsid protein structure of a novel norovirus GII.17 variant isolated in China was extensively characterized. The data would be helpful not only for the epidemiology study, but also for the diagnostic tool development and effective vaccine design in the future.

HIV vaccine research: the challenge and the way forward.

Human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is a worldwide epidemic, with over 35 million people infected currently. Therefore, the development of a safe and effective HIV-1 vaccine is on top of the global health priority. In the past few years, there have been many promising advances in the prevention of HIV/AIDS, among which the RV144 Thai trial has been encouraging and suggests optimization of the current vaccine strategies or search for novel strategies. Here we reviewed the brief history of HIV-1 vaccine, analyzed key challenges existing now, and illustrated future research priority/directions for a therapeutic or prophylactic HIV-1 vaccine, with the hope of accelerating the speed of vaccine development. We believe that an effective HIV-1 vaccine, together with other prevention approaches, will bring an end to this epidemic in the near future.

Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification.

Rotaviruses, noroviruses and astroviruses are the major viral pathogens leading to diarrhea worldwide. Epidemiological investigations of outbreaks associated with these viruses have been impeded by the lack of methods for quick diagnosis and detection. In the current study, a multiplex real-time nucleic acid sequence-based amplification (RT-NASBA) system was developed for the simultaneous detection of rotavirus A/norovirus genogroup II/astrovirus. The specificity and sensitivity of the assay were compared with multiplex RT-PCR. The results showed that the multiplex RT-NASBA assay was established successfully, and robust signals could be observed in 10 minutes with high specificity. The limit of detection of the multiplex RT-NASBA assay was 7, 100, and 200 copies per reaction for rotavirus A, norovirus genogroup II, and astrovirus, respectively. The assay was thus 10 to 100 times more sensitive than multiplex RT-PCR. Clinical evaluation indicated that the assay was 100% concordant with multiplex RT-PCR and was reliable for the detection of both single infections and multiple infections in stool samples. To the best of our knowledge, this is the first multiplex RT-NASBA assay established for the detection of three major diarrhea-causing viruses. This assay provides a valuable platform for the rapid, specific, sensitive and simultaneous diagnosis of these pathogens, especially in resource-limited countries where expensive thermocycling equipment is not available.

Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

[Rapid detection of novel avian influenza virus subtype H7N9 by multiplex real-time RT-PCR].

In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.

[Epidemiological features on 3 important viral diarrhea among children in Zhuhai during winter and spring].

OBJECTIVE: To explore the epidemiological characteristics of viral diarrhea of norovirus (NV), sapovirus (SV) and astrovirus (AstV) among children in Zhuhai during winter and spring. METHODS: Stool specimens were collected from children with viral diarrhea in Maternal and Child Health Hospital of Zhuhai from November 21, 2009 to April 3, 2010. Nucleic acid of NV, SV and AstV from negative specimens of rotavirus and adenovirus were detected by using Reverse transcription-polymerase chain reaction (RT-PCR), and the types of positive samples of NV were also classified at the same time. RESULTS: The total detection rate of the three viruses is 21.49 percent, the highest detection rate is 29.05% in December 2009, the lowest detection rate is 12.20% in February 2010, 87.96% of positive specimens were from children patients aged from 0 to 30 months. The season detection rate of NV, SV and AstV are 14.70%, 2.75% and 4.04% respectively. There were significant differences of NV and SV detection rates in every month of the season, whereas the AstV detection rate was comparatively stable. The highest detection rate of NV is 34.09% in children patients aged from 12 to 18 months, the highest SV detection rate is 12.5% in children patients aged from 60 to 120 months, and the highest AstV detection rate is 16.67% in children patients aged from 24 to 30 months. All the NV were belong to G II genogroup. CONCLUSIONS: NV is one of the main pathogens causing viral diarrhea among children in Zhuhai during winter and spring, SV and AstV are also important pathogens. So we should strengthen the monitoring of viral diarrhea caused by NV, SV and AstV in infants and young children.

[HAND system-based four multiplex RT-PCR for simultaneous detection of four diarrhea viruses].

OBJECTIVE: To establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus. METHODS: Primers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically. RESULTS: The 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively. CONCLUSION: The established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α-thalassemia in maternal plasma.

Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.

[Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning].

OBJECTIVE: To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria. METHODS: Type-specific PCR primers labeled with biotin and oligonucleotide probes were designed according to the conservative genes of 9 major foodborne bacteria Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7 (Stx1 and Stx2), Shigella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus. A one-tube multiplex PCR system for simultaneous amplification of these bacteria was established, and the DNA probes were spotted and immobilized in the wells of the plate in 5x5 array format. Stable hybridization system between PCR products and oligonucleotide probes in the microwell was established after condition optimization. Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products. RESULTS: Twenty standard bacteria strains were used to validate the 96 microwell plate DNA diagnostic chip and highly specific and stable experiment results were obtained. Using this chip assay, the causal pathogen Staphylococcus aureus was identified within 12 h after the sampling from an incident of food poisoning, and the result was consistent with that obtained using conventional bacterial culture and biochemical identification. CONCLUSION: The novel 96 microwell plate DNA diagnostic chip allows rapid, accurate, automated and high-throughput bacterial detection and is especially valuable for quick response to such public health emergencies as food poisoning.

Direct fluorescence detection of point mutations in human genomic DNA using microbead-based ligase chain reaction.

This report has described a convenient genotyping method capable of detecting point mutations directly in human genomic DNA based on the combination of ligase chain reaction (LCR) and microbead-enrichment technique. LCR primers, including a biotin-labeled common primer and two fluorescence-labeled allele-specific primers, are designed for two alleles of a mutated site. When genomic DNA carries the mutated site, the common primer and allele-specific primer are ligated to form exponential amplified biotin-labeled fluorescence ligation products. These ligated products are enriched by streptavidin-coated microbeads, and genotypes are identified conveniently according to the fluorescence color of microbeads using fluorescent microscopy. Due to amplification of LCR process and enrichment of microbeads, the detection limit of the proposed method is as low as 10(-15)mol/L templates. The method provides a convenient and simple strategy to detect point mutation directly in human genome. We have confirmed the efficiency of this approach with the identification of beta-globin gene point mutation, which results in the reduced production of globin in an inherited hemoglobin disorder thalassemia disease.

[One-step multiplex RT-PCR for rapid screening of type A, B and novel A (H1N1) influenza viruses].

OBJECTIVE: To developed a multiplex RT-PCR assay for simultaneous screening of type A, B and novel A (H1N1) influenza viruses. METHODS: Two pairs of universal primers in were designed for amplifying the M gene and NS gene of type A and B influenza viruses, respectively. A pair of specific primers of HA gene was designed to detect novel A (H1N1) influenza virus. A one-step method was used to establish the multiplex RT-PCR system. A blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. The clinical practicability and efficacy of this assay was also evaluated. RESULTS: The RT-PCR products were analyzed using agarose gel electrophoresis, which yielded distinct bands of the target fragments without non-specific reactions, suggesting the high efficiency and specificity of the multiplex RT-PCR. Blinded study of 50 samples demonstrated a concordance rate of 100%. CONCLUSION: This multiplex RT-PCR assay allows one-step simultaneous detection of type A, B and novel A (H1N1) influenza viruses rapidly and accurately, and provides a valuable low-cost screening technique for influenza epidemic monitoring and early diagnosis.

[A community-based genetic screening of large-scale population and prenatal diagnosis for alpha and beta thalassemia in Zhuhai city of Guangdong province].

OBJECTIVE: To describe a community-based model for prevention and control of severe alpha and beta thalassemias in Zhuhai city of Guangdong province. METHODS: Couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled in this prospective screening program, which was supported by the two-level network composed of 6 local hospitals for testing thalassemias and follow-up for genetic counseling. A conventional heterozygote screening strategy was used to determine alpha and beta thalassemia traits in women and their partners according to the standard procedures of hematological phenotype analysis. Then confirmative diagnosis of alpha and beta thalassemia was performed on those couples suspected at-risk for severe thalassemia by using the PCR-based molecular diagnostic assays. The couples at-risk for severe thalassemia were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus. RESULTS: During the period between January 1998 and December 2005, the screened records included 85522 young females and their partners for premarital screening and 10439 pregnant women for prenatal screening, with 71.38% coverage of total population recorded in this city for premarital screening. Six thousands five hundreds and sixty-three individuals in total were found to be the carriers of thalassemias, with 4312 for alpha thalassemia (4.5%) and 2251 for beta thalassemia (2.3%), respectively. One hundred and forty-eight couples were diagnosed to be at-risk for thalassemias, including 103 for alpha thalassemia and 45 for beta thalassemia, respectively. Successful prenatal diagnosis was made for 142 (98 for alpha thalassemia and 44 for beta thalassemia) out of 148 (95.9%) pregnancies at-risk for severe thalassemias. Twenty-three cases of hydrops fetalis, 4 of Hb H diseases and 14 of beta thalassemia were identified. All 41 pregnancies with affected fetuses were voluntarily terminated. Thus, this has led to a marked decrease of severe thalassemia syndrome since the program started. CONCLUSION: We presented the first community-based prospective screening program in China for control of alpha and beta thalassemia in Zhuhai city with a population of 1.29 million through premarital or prenatal screening. This model could be used for control of thalassemias and other hemoglobinopathies in other regions of China and also in other developing countries.