重组酶聚合酶扩增结合CRISPR-Cas12a快速检测十足目虹彩病毒1方法的建立

[背景] 十足目虹彩病毒1（Decapod Iridescent Virus 1,DIV1）可感染南美白对虾、中国对虾和日本对虾等,是危害对虾养殖业的主要病毒之一。当前高效、快速、简便地检测对虾是否感染DIV1是减少其发生和危害的重要途径。[目的] 将成簇的规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR）及其相关蛋白12a （CRISPR Associated Protein 12a,Cas12a）,即CRISPR-Cas12a系统,与重组酶聚合酶扩增（Recombinase Polymerase Amplification,RPA）技术相结合,建立快速检测DIV1的方法（RPA-Cas12a）,并探讨该方法在实际样本检验中的应用价值。[方法] 通过提取DIV1 DNA,设计其RPA引物、crRNA及报告探针,优化并建立RPA结合CRISPR-Cas12a的快速检测方法,进一步分析该方法检测DIV1的灵敏度与特异性,并比较建立的方法与qPCR法的一致性。[结果] 建立的RPA-Cas12a快速检测方法可在40 min内实现对虾样本DNA中DIV1的检测,检测限为10 copies/reaction。用该方法分别对对虾白斑综合征病毒（White Spot Syndrome Virus,WSSV）、传染性皮下及造血组织坏死病毒（Infectious Hypodermal and Hematopoietic Necrosis Virus,IHHNV）、虾肝肠胞虫（Enterocytozoon Hepatopenaei,EHP）及DIV1进行检测,结果仅DIV1发生特异性反应,而WSSV、IHHNV和EHP的检测结果均为阴性。采用RPA-Cas12a检测61份实际样本,结果均与qPCR检测法的阳性检出率一致。[结论] 建立的RPA-Cas12a方法检测十足目虹彩病毒1具有快速、简便、灵敏度高且特异性强的特点,为十足目虹彩病毒1的快速检测提供了新的工具。     

检测蜜蜂急性麻痹病毒TaqMan实时荧光RT-PCR方法的建立和初步应用

[背景] 蜜蜂急性麻痹病毒（Acute Bee Paralysis Virus,ABPV）是一种高毒力的蜜蜂病毒,可以引起蜜蜂的大批死亡和蜂群衰竭。[目的] 建立一种快速、灵敏的ABPV实时荧光RT-PCR检测方法。[方法] 根据ABPV衣壳蛋白基因保守序列设计引物和探针,通过对引物、探针浓度和退火温度等反应条件进行优化,建立基于TaqMan探针检测ABPV的实时荧光RT-PCR方法,并对方法的灵敏性、特异性和稳定性进行验证。[结果] ABPV实时荧光RT-PCR检测方法在9.8×10¹-9.8×10⁸ copies/μL之间呈现良好的线性关系,线性相关系数R²为0.998,扩增效率为103.8%。该方法的检测灵敏度为9.8 copies/μL;对其他蜜蜂病毒不发生交叉反应,具有良好的特异性;重复性试验结果显示组内和组间的变异系数分别为0.19%-0.80%和0.57%-1.07%,重复性良好。对2018年-2019年在福建地区采集的70份蜜蜂样品进行ABPV检测,阳性率为2.86%。[结论] 建立的ABPV实时荧光RT-PCR检测方法能用于该病的实验室检测、流行病学调查和疫情监测。     

酶促重组等温扩增实时荧光法快速检测肺炎支原体方法的建立及应用北大核心CSCD

利用酶促重组等温扩增(enzymatic recombinase amplification,ERA)技术建立快速检测肺炎支原体的实时荧光检测方法。针对肺炎支原体P1基因设计特异性引物和探针,优化反应条件,分析其敏感性和特异性,并对临床样本进行验证。ERA实时荧光法在25-40℃均具有扩增能力,在35℃条件下对肺炎支原体的扩增效果最好,20 min内可完成扩增;该法对肺炎支原体的检出限为10^(3) copies/μL;并对其他6种呼吸道病原体进行检测,均无扩增曲线产生,有较好的特异性;以荧光定量PCR法检测结果为标准,ERA实时荧光法对34份临床样本的检测结果的诊断敏感度为96.15%、特异度为100%、阳性预测值为100%、阴性预测值为88.89%。本研究构建的ERA实时荧光法可以快速简单、灵敏和特异地检测出肺炎支原体,满足现场检测的需求。     

多重定量PCR检测PDX模型小鼠细胞浸润比例的构建

摘要 目的：建立一种快速、灵敏的方法检测人源异种移植模型(Patient-Derived tumor Xenograft,PDX)中小鼠细胞的浸润比例,以确保PDX模型的保真性。方法：选择人和小鼠的特异性基因PSMB2、Ren 2的部分区段设计引物和探针,利用多重荧光定量PCR（TaqMan探针法）在单管中检测PDX模型中小鼠-人细胞的相对量。另外,构建了不同浸润比例的标准品作为阳性对照,根据标准曲线进行样本比例计算。结果：建立了一种多重荧光定量PCR检测PDX模型小鼠细胞浸润比例的检测方案,该方案显示,PDX模型中不同比例的人鼠混合样本与△Ct值（Ct（小鼠）-Ct（人））之间线性关系良好,相关系数r²为0.9998。利用该方法对14个样本进行检测,结果表明在绝大部分样本中小鼠细胞的比例低于30.00%,平均小鼠细胞比例为13.38%。结论：本研究建立的多重定量PCR检测PDX模型中小鼠细胞浸润比例的方法能够根据△Ct值快速、准确推断PDX模型中人、小鼠细胞的比例。该方法操作简单、耗时短、结果可靠,是一种快速、灵敏的PDX模型的质控方案。     

实时荧光重组酶聚合酶扩增快速检测临床常见曲霉菌方法的建立

目的:针对曲霉菌属转录间隔区ITS1设计引物、探针,利用实时荧光重组酶聚合酶扩增(Real-time RPA)技术建立一种快速、准确、经济的临床常见曲霉菌检测鉴定方法。方法:利用建立的实时荧光重组酶聚合酶扩增体系对标准菌株及临床标本提取的DNA进行扩增,验证该方法的性能。结果:本研究针对曲霉菌属转录间隔区ITS1设计引物、探针利用RPA试剂盒(荧光型)建立了Real-time RPA扩增体系,在15分钟内即可检测出临床常见的四种曲霉菌;特异性试验结果显示反应体系只对烟曲霉、黄曲霉、土曲霉、黑曲霉四种曲霉呈现出明显的扩增曲线,而其它细菌和真菌均无扩增曲线。灵敏性试验显示最低检出限为10-3 ng/μL。临床验证试验的12份曲霉菌均有较高的扩增效应。结论:本研究建立的Real-time RPA方法能快速、特异、灵敏地检出烟曲霉、黄曲霉、土曲霉、黑曲霉等临床常见曲霉菌,为曲霉菌的快速、现场检测提供了一种新的思路。     

转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

转基因玉米双抗12-5具有良好的抗虫性和除草剂耐受性,是我国第一批获得安全证书的转基因玉米之一,具有广阔的应用前景。本研究利用重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)建立转基因玉米双抗12-5的现场快速检测方法。针对转基因玉米双抗12-5的转化体特异性序列片段,设计引物和探针,通过引物筛选实验得到最佳引物与探针组合。荧光RPA扩增结果可以在蓝光下直接进行可视化分析。结果表明,建立的转基因玉米双抗12-5可视化检测体系特异性强,检测灵敏度达到10拷贝。进一步研究发现RPA的扩增体系对温度有很强的适应性,样品在34℃～46℃之间都能得到扩增,据此,本研究利用市面上常见的自发热暖贴代替常规的加热仪器来激发RPA。结果表明,自发热暖贴满足RPA扩增体系对温度的需要。最终,本研究将自发热暖贴加热法与RPA可视化检测体系结合,对转基因玉米双抗12-5进行现场检测,并与qPCR方法检测结果作比较,检测结果表明,本研究建立的现场可视化检测方法与qPCR方法检测结果一致,并且可视化检测方法时间短,检测结果清晰易分辨。本研究建立的转基因玉米...     

支原体检测多重定量PCR方法的建立及应用

目的:建立一种快速、准确的方法检测支原体,这不仅可以有效地减少和预防支原体污染,还能为科研工作者提供一定的指导价值。方法:利用荧光定量PCR(TaqMan探针法)检测支原体,反应体系中同时存在标记两种颜色TaqMan探针及相关引物,分别检测支原体DNA和参考基因模板。根据支原体16S核糖体RNA保守区和参考基因TOP3A保守区设计引物和探针。通过对引物浓度、探针浓度和退火温度等反应条件的优化,建立了TaqMan探针多重定量PCR方法,并对该方法的特异性、敏感性和重复性进行了验证。结果:建立的双色荧光探针定量PCR方法的标准曲线相关系数r2和扩增效率分别为0.995和113.36%;该方法最低检测限为10 copies/μL;组内及组间变异系数均小于1%,证明该检测方法高效。利用该方法对随机挑选90例细胞抽提DNA样本进行检测,结果有60例为支原体阳性样本,阳性率67%,阳性率与相关研究报道一致。检测3个细胞培养上清样本,结果 1例支原体阳性,2例支原体阴性。从检测的样本中随机选择3个阳性样本及2个阴性样本使用普通PCR支原体检测试剂盒检测,结果一致;将其测序,测序结果比对正确。结论:本研究建立的多重定量PCR支原体检测方法能够应用于细胞抽提DNA及细胞培养上清的支原体检测,可以实现高效、快速检测支原体污染。     

多酶恒温扩增核酸试纸条法快速检测肺炎支原体方法的建立及应用

【背景】肺炎支原体是导致儿童和青少年呼吸道感染的重要病原体,长期以来由于其临床表现不特异而容易错过最佳治疗时期。【目的】结合多酶恒温扩增(multienzyme isothermal rapid amplification,MIRA)技术和核酸试纸条建立一种快速检测肺炎支原体的方法。【方法】以肺炎支原体社区获得性肺炎呼吸窘迫综合征(community acquired respiratory distress syndrome, CARDS)毒素编码基因为靶基因设计引物和探针,对反应体系的温度、时间等进行优化,评估其敏感性,通过检测肺炎支原体和其余7种病原体分析其特异性,并对35份临床样本进行验证。【结果】MIRA核酸试纸条法在37℃条件下,15 min内便可完成对肺炎支原体的检测,最低检出限为10 copies/μL;除肺炎支原体外,其余7种病原体均不能扩增,特异性较好。以实时荧光PCR检测为标准,MIRA核酸试纸条法对35份临床样本检测后的诊断特异度为100.00%、灵敏度为96.15%、阴性预测值为90.00%、阳性预测值为100.00%。【结论】本研究建立了MIRA核酸试纸条法...     

TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

咽拭子快速培养在肺炎支原体感染中的临床应用价值

目的:探讨咽拭子快速培养在肺炎支原体感染中的临床应用价值。方法:收集2014年2月~2016年2月期间我院收治的呼吸道感染患儿220例,用肺炎支原体专用液体培养基进行肺炎支原体快速培养,用胶体金法检测肺炎支原体MP-Ig M。比较两种方法的阳性率。结果:咽拭子培养快速培养阳性率与血清MP-Ig M检测阳性率比较,差异无统计学意义(P0.05)。MP-Ig检测显示,≤1岁阳性率最低,其阳性率随年龄增加不断增高(P0.05)。肺炎支原体咽拭子培养显示,≤1岁阳性率最高,2~8岁最低(P0.05)。病程≤7 d患者肺炎支原体咽拭子培养阳性率(34.21%)显著高于肺炎支原体MP-Ig检测阳性率(14.04%)(P0.05)。病程7 d患者肺炎支原体咽拭子培养阳性率(11.32%)显著低于肺炎支原体MP-Ig检测阳性率(52.83%)(P0.05)。肺炎支原体咽拭子培养的灵敏度性以及特异性显著高于肺炎支原体MP-Ig检测,差异具有统计学意义(P0.05)。结论:咽拭子快速培养对肺炎支原体感染的早期诊断有一定临床应用价值,方法简单,无创伤,值得临床进一步研究和应用。     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

基于LAMP技术建立解脲支原体基因试纸条检测方法

目的建立一种灵敏度高、特异性强、检测速度快的方法检测解脲支原体。方法基于环介导恒温扩增技术（LAMP）,根据解脲支原体序列特征设计3对引物进行解脲支原体DNA切口酶核酸恒温扩增,扩增过程在一对引物中标记生物素,随着扩增的进行生物素直接引入扩增片段中,扩增结束后产物在密闭装置中进行免疫试纸条显色反应,根据显色卡的颜色判定结果的阴阳性。结果该技术检测解脲支原体较实时荧光PCR技术灵敏度要高10倍以上,其它病原体检测均阴性该方法特异性与实时荧光PCR技术相当。结论恒温扩增联合试纸条技术检测解脲支原体具有较高的敏感性和特异性,检测速度快,适合各医院开展。     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.     

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

Background

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV.

Methods

Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens.

Results

RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%.

Conclusion

IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

快速检测NDM-1基因的环介导恒温扩增技术的建立与评价

基于DNA环介导恒温扩增技术 (Loop-mediated isothermal amplification,LAMP),探索建立一种应用于NDM-1基因 (New Metallo-β-Lactamase-1 Gene,NDM-1) 的快速检测方法,以适应临床实验室等的检测需求。利用LAMP技术,以NDM-1基因为靶序列,设计4组LAMP引物,并筛选最优引物组,建立LAMP反应体系与条件,进行灵敏度和特异性实验。结果表明整个检测过程仅需1 h,即可通过肉眼直接目测实验结果。在灵敏度试验中,NDM-1基因的最低检测限为6 拷贝/反应。在特异性实验中,以4株病原菌 (肺炎克雷伯氏菌、大肠埃希氏菌、金黄色葡萄球菌、肺炎链球菌) 以及肠道菌群元基因组DNA、土壤菌群元基因组DNA为模板对NDM-1基因进行检测,结果显示均没有发生非特异性扩增反应。文中建立的LAMP检测方法能够快速检测NDM-1基因,且可直接观察到实验结果,实现了检测结果的可视化。具有操作简单安全、检测灵敏度高、特异性高的特点,能够满足基层实验室、应急检测或现场监测等方面的使用需求,具有良好的应用价值。     

Sensitive and semiquantitative detection of soil-transmitted helminth infection in stool using a recombinase polymerase amplification-based assay

BackgroundSoil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs.MethodologyWe developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples.Principal findingsEach RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals.ConclusionsSemi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity.