Advancing public health preparedness:Establishment of Nipah virus molecular diagnostic test at the National Reference Laboratory,Sri Lanka

Objective:To establish Nipah virus diagnostic capabilities at the National Reference Laboratory in Sri Lanka using the NIV Pune real-time PCR kit.Methods:Strict safety precautions were adhered during testing due to the high pathogenicity of the Nipah virus,with all diagnostics conducted in a BSL2+laboratory at the Medical Research Institute in Sri Lanka.RNA extraction was performed using the QIAamp Viral RNA Mini kit.The NIV Pune in-house real-time PCR kit was employed,following established primer/probe sequences and controls.The assay was validated using the Rotor-Gene Q Series Real-time PCR platform.Results:The validation run of the Nipah virus real-time PCR test demonstrated robust performance,with positive controls consistently detecting Nipah RNA at a Ct value of 21.50±0.01.Negative controls confirmed assay specificity with an external negative control which was also used as an extraction control and showed no interference.The internal control exhibited stable behavior,enhancing confidence in PCR results.The qPCR analysis graph illustrated the successful detection of internal and positive controls,validating the reliability of the assay.Conclusions:Establishing Nipah virus diagnostic capabilities in Sri Lanka signifies a proactive and collaborative response to the persistent global health threat.

Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification

We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.

COVID 19 Diagnostic Tests: A Study of 12,270 Patients to Determine Which Test Offers the Most Beneficial Results

COVID 19 has caused capitulation from healthcare entities all over the world. First described in Hubei, China, the virus has spread to 185 countries, showing little signs of eradication or eradication. There does not exist a medical treatment regimen or a vaccine to address COVID 19 definitively. The best response, to date, has been early diagnosis and immediate isolation or quarantine of the patient, with supportive care. As medical institutions all around the world struggle to keep up with this pandemic, there is not a consensus amongst medical professionals in the rapid diagnosis of this disease entity. Purpose: The purpose of our study was to review the literature and establish a test, or tests, that would aid the clinician in attaining a swift, yet accurate diagnosis. Methods: We searched PubMed and Google scholar and reviewed 32 articles. Keyword searches consisted of COVID 19, pandemic, diagnoses, diagnostic testing, pandemic amongst others. We compared the data obtained from these studies in an effort to find the best diagnostic test. Results: There were a total of 12,270 patients that were in our study [1]-[32]. This is the largest study to date in the literature addressing diagnosis of COVID 19. Fever, cough and fatigue, in that respective order were the most common clinical symptoms. Laboratory findings consisted of leukopenia, elevated erythrocyte sedimentation (ESR) and elevated C-reactive protein, CRP. The gold standard test described in multiple studies was the RT-PCR. Serum assays of IgM and IgG were also drawn and found to be accurate in 93% of the time. CT Chest was both sensitive and specific, 90% and 86%. This diagnostic imaging was even more successful when coupled with clinical symptoms and approaching days 7 - 12 since the onset of clinical symptoms. Discussion: This is the largest study compiled to address diagnostic testing in COVID 19 patients. The patient population is spread vastly around the world, with access to many reported tests limited in certain countries. Given the significant sensitivity and specificity of diagnostic imaging, in the setting of clinical symptoms, we recommend patient undergo CT Chest in the face of COVID 19 exposure and clinical symptoms. While RT-PCR, IgM-IgG assays are beneficial, isolation, treatment, and possible quarantine of presumptive positive COVID 19 patients (based upon clinical symptoms and imaging) should not be delayed, for fear of increased infectivity and further risk to society at large.

Comparison of Two Molecular Diagnostic Tests for COVID-19: Abbott RealTime SARS-CoV-2 and Allplex™2019-nCoV, in the Epidemic Context in Senegal

Background: The persistence of the rapid spread of the COVID-19 pandemic is linked to the appearance of several variants of SARS-CoV2 with an impact on biological diagnosis, treatment and vaccination. The United States Food and Drug Administration (FDA) has granted several SARS-CoV-2 detection tests Emergency Use Authorization (EUA) for diagnosis and better epidemiological surveillance. Thus, multiple RT-PCR tests have been developed and brought to market in order to meet the urgent need for the diagnosis of COVID-19. However, comparative data between these tests in clinical laboratories are scarcely available to assess their performance. Objective: To compare two molecular methods for detecting SARS-CoV-2: the RT-PCR, Allplex™2019-nCoV tests on CFX96 Bio-Rad and the Abbott m2000sp/rt RealTime SARS-CoV-2. Materials and Methods: Nasopharyngeal and oropharyngeal swabs were taken from patients to diagnose SARS-CoV-2 infection. For each sample, we searched for the virus with two different RT-PCR tests: 1) first on Abbott m2000 SARS-CoV-2 targeting the N and RdRp genes, 2) then on Allplex™2019-nCoV Assay looking for the E, N and RdRp genes. Results: Percentages of the agreement were calculated. A total of 100 samples that tested negative and 90 positives on Abbott m2000 SARS-CoV-2 were retested on Allplex™2019-nCoV. Overall agreement was 74.74% on all samples. The specific agreement was 84% and 64.4% respectively for negative and positive samples with the RealTime SARS-CoV-2 test. A positive correlation (r2 = 0.63;p Conclusion: Our results showed good overall agreement between RT-PCR, Allplex™2019-nCoV and Abbott RealTime SARS-CoV-2 tests in the diagnosis of COVID-19. As the concordance is low for small viremias, the RT-PCR Allplex™2019-nCoV Assay would be better indicated during the acute and symptomatic phase of the disease.

基于TashHN基因的牛环形泰勒虫病PCR诊断方法的建立

根据Genbank已发表的环形泰勒虫TashHN基因序列设计合成引物,以此建立基于环形泰勒虫TashHN基因的PCR检测方法,对该方法的特异性、敏感性、稳定性进行验证,并用建立的方法对宁夏固原地区各县共计135份牛血样进行检测。结果表明:建立的基于TashHN的PCR检测方法与中华泰勒虫、东方泰勒虫、牛巴贝斯虫、双芽巴贝斯虫均无交叉反应;可检测的最低模板浓度为1.64×10^(2) copies/μL;环形泰勒虫的DNA在-20℃冰箱放置8个月后仍可检测出目的条带;该方法利用已有文献中的PCR方法进行一致性检测,从135份牛血样中检测出5份阳性,说明该方法准确可靠。因此,试验建立的基于环形泰勒虫TashHN基因的PCR检测方法具有特异性强、敏感性高、稳定性强的特点,适用于临床上环形泰勒虫早期感染检测以及流行病学的调查。

分析酶联免疫吸附法及RT-PCR法检验血液标本内乙肝病毒的价值

目的探讨酶联免疫吸附法及RT-PCR法检验血液标本内乙肝病毒的价值。方法将2022年1月—2023年10月在本院治疗的78例疑似乙肝患者作为研究对象,根据检测方法不同分为A组和B组,每组39例。两组使用酶联免疫吸附法(ELISA)、实时荧光定量PCR(RT-PCR)检测乙肝病毒(HBV)血清标志物、HBV-DNA含量。其中,A组采用单纯ELISA检验,B组采用单纯RT-PCR检验。以临床最终诊断结果作为金标准,分析两组检测结果,对比诊断效能。结果A组中,HbeAg阳性率39.74%(31/78),HBsAg阳性率58.97%(46/78),HbcAb阳性率51.28%(40/78),HbeAb阳性率56.41%(44/78),HbsAb阳性率61.54%(48/78),大三阳的阳性率39.74%(31/78),小三阳的阳性率51.28%(40/78);B组中,HBV-DNA阳性率80.77%(63/78);A组对HBV的诊断敏感度、准确度、阴性预测值均低于B组(P<0.05),但两组特异度、阳性预测值相当(P>0.05)。结论RT-PCR法检验血液标本内HBV的价值更高,对HBV的敏感度及准确度高,整体诊断效能更高。

实时荧光定量PCR技术在布鲁氏菌检验中的应用

目的 分析细菌布鲁氏菌的临床检验中应用实时荧光定量多聚酶链式反应(polymerase chain reaction,PCR)技术的效果与效能。方法 非随机选取山东省枣庄市立医院于2020年7月—2023年7月收治的经血培养确诊为细菌感染的97例患者作为研究对象,所有受检对象均进行常规PCR检查与实时荧光定量PCR技术检查,以血培养结果为金标准,对比不同PCR技术对布鲁氏菌的诊断结果和诊断效能。结果 在97例细菌感染患者中金标准检出布鲁氏菌感染66例,常规PCR技术检出布鲁氏菌感染55例,实时荧光定量PCR技术检出布鲁氏菌感染63例。实时荧光定量PCR技术对布鲁氏菌感染的诊断准确度为94.85%(92/97)、灵敏度为93.94%(62/66)、阴性预测值为88.24%(30/34),均高于常规PCR技术的准确度78.35%(76/97)、灵敏度75.76%(50/66)、阴性预测值61.90%(21/42),差异有统计学意义(χ^(2)=11.370、8.486、6.718,P均<0.05),两种检验方式的特异度及阳性预测值差异不明显,差异无统计学意义(P均>0.05)。结论 应用实时荧光定量PCR技术检测细菌布鲁氏菌效果较好,有利于及时发现病情进展,并为及时治疗提供有效依据。

实时荧光PCR技术在手足口病检测中的应用价值分析

目的 探析实时荧光聚合酶链式反应(polymerase chain reaction,PCR)技术在手足口病检测中的应用价值。方法 非随机选取2021年1月—2023年11月高唐县疾病预防控制中心收治的100例疑似感染手足口病患儿为研究对象,均进行实时荧光PCR检查、DNA测序技术检查,以病毒分离培养结果为金标准,对比两种技术的诊断情况。结果 病毒分离培养结果显示100例疑似患儿中有73例确诊。实时荧光PCR技术诊断效能灵敏度97.26%(71/73)、特异度88.89%(24/27)、准确度95.00%(95/100)、阳性预测值95.95%(71/74)、阴性预测值92.31%(24/26)明显高于DNA测序技术的72.60%(53/73)、55.56%(15/27)、68.00%(68/100)、81.54%(53/65)、42.86%(15/35),差异有统计学意义(χ^(2)=17.340、7.477、24.175、7.462、15.821,P均<0.05)。实时荧光PCR技术的病毒亚型检出率高于DNA测序技术,差异有统计学意义(P均<0.05)。结论 在手足口病检测中,实时荧光PCR技术的应用价值高于DNA测序技术,更利于为后续治疗提供可靠参考依据。

齿瓣石斛的位点特异性PCR鉴别

目的 设计出齿瓣石斛的位点特异性鉴别引物 ,仅通过PCR就能完成对齿瓣石斛真伪进行准确鉴别。方法 根据齿瓣石斛及其他枫斗类、黄草类石斛的rDNAITS序列数据库 ,设计了齿瓣石斛的位点特异性PCR鉴别引物JB Chiban 0 1S和JB Chiban 0 1X。然后 ,对 38种石斛属植物模板DNA进行了PCR扩增 ,阳性者即为齿瓣石斛正品。结果 当退火温度设定为 5 8℃时 ,只有齿瓣石斛的模板DNA能被扩增出来 ,而其他的 37种石斛属植物均为阴性。该鉴别反应重复性好 ,已在鉴别齿瓣石斛时发挥重要作用。结论 运用位点特异性鉴别引物能成功地对齿瓣石斛进行PCR鉴别 ,与DNA测序鉴别方法相比 ,位点特异性PCR具有高效、准确、简便、省时等优点。

中药材龟甲及原动物的高特异性PCR鉴定研究

目的:建立一种简便、实用的龟甲药材DNA 分子鉴定方法。方法:根据22 种亚洲产龟类的线粒体12SrRNA 基因片段序列,设计一对专用于鉴定中药材龟甲原动物乌龟的鉴别引物,用该对引物扩增从乌龟和其他18 种龟共48 个样品的DNA 模板。结果:在72℃的复性温度下进行PCR,4 个乌龟的模板DNA 均得到约180 bp 的阳性扩增带,而其他各龟的模板DNA,在同样条件下无扩增产物,用这对鉴别引物经一次PCR 反应便可准确地鉴定受试原动物是否为乌龟。同法对江苏省药品检验所提供的17 块样品龟甲进行了鉴定,结果表明只有4 块样品为正品,其余皆为伪品,与性状鉴定和DNA序列分析鉴定结果完全一致。结论:所设计的鉴别引物对乌龟有高度特异性。

丙型肝炎病毒(HCV)荧光PCR(F-PCR)试剂盒的研制及与免疫学方法的比较

目的:用荧光PCR（Fluorescence PCR,F-PCR）方法研制丙型肝炎病毒（Hepatitis C Virus,HCV）RNA检测试剂盒,通过临床验证,考核其性能,并与免疫学方法比较。方法:F-PCR是PCR和荧光探针杂交技术结合所产生的PCR方法。由于采用完全闭管荧光检测,避免了PCR后处理导致的假阳性污染;又采用了荧光检测技术,可以提高检测的灵敏度。结果:设计合成了HLV F-PCR诊断试剂盒。检测了 512份临床血清标本,以美国 Abbott公司的HCV ELISA诊断试剂盒和美国Biotronics公司的HCV荧光RT-PCR诊断试剂盒（B-PCR）为对照。阳性率30.5％,灵敏度97.3％,特异性98.1％。结论:F-PC的灵敏度显著优于ELISA,也高于B-PCR;三者的特异性无统计学差异。F-PCR试剂盒可以检测HCV的真实感染情况,对于临床诊断和疗效考察有一定的指导意义。

牙鲆淋巴囊肿病的PCR诊断方法研究

以中国养殖牙鲆 (Paralichthysolivaceus)淋巴囊肿病毒 (Lymphocystisdiseasevirus,LCDVcn)主要衣壳蛋白 (majorcapsidprotein ,MCP)基因的中间保守序列为目标基因 ,设计了一对特异性引物。该引物可扩增出 172bp的病毒DNA片段 ,其最小DNA检出量为 0 0 183ng。用PCR法从人工感染淋巴囊肿病毒 3天的牙鲆血、鳃、肝、脾、肠、胃及自然发病牙鲆的肿瘤中 ,分别检测到了LCDV的存在。本实验结果证明 ,PCR法对于早期检测LCDV是十分有效的。

鹿类中药材的位点特异性PCR鉴定研究

目的 建立一种简便、准确的鹿类中药材鹿茸、鹿鞭、鹿筋、鹿胎的DNA分子标记鉴定方法。方法 在对鹿类中药材的正品原动物梅花鹿、马鹿及其混伪品原动物的Cytb基因全序列分析的基础上 ,设计了一对专用于鉴定正品鹿类药材的位点特异性鉴别引物ILu0 1 L和ILu0 1 H。结果 在 6 4℃的复性温度下 ,用鉴别引物对原动物样品进行鉴别PCR ,仅正品能得到约 36 5bp阳性扩增带 ;对鹿茸、鹿鞭及鹿筋正、伪品药材进行PCR鉴定 ,结果表明 :3批鹿茸仅一批为正品 ,2批鹿鞭皆为伪品 ,鹿筋正、伪品药材PCR鉴定与形态鉴定结果一致。随机选取 2枚鹿茸及一个原动物做Cytb基因片段序列分析 ,其结果与PCR鉴定完全一致。结论 对市售鹿类商品药材需加强质量监督和管理。所设计的鉴别引物对梅花鹿、马鹿有高度特异性 ,可应用于以其为原动物的鹿类中药材的鉴定。

象耳豆根结线虫的PCR鉴定和检测方法(英文)

象耳豆根结线虫是一种在中国具有潜在经济重要性的农作物病原物。为提供有助于控制象耳豆根结线虫传播扩散的方法,研制了该线虫的快速PCR鉴定和检测法。该方法PCR引物的扩增目标为rDNA-IGS2区域,其设计依据象耳豆根结线虫与南方、爪哇、花生和北方根结线虫在该区域核酸序列的差异。通过对6种近似根结线虫的不同地理群体及自然土壤线虫群体的测试,验证了设计的PCR引物针对象耳豆根结线虫的特异性和可靠性。本方法具有快速灵敏的特点,可用于象耳豆根结线虫单条线虫的直接鉴定以及混合土壤线虫群体中象耳豆根结线虫的检测。

金钗石斛的位点特异性PCR鉴别研究

目的建立一种简便、实用的金钗石斛的DNA分子鉴别方法。方法根据金钗石斛及其他黄草类、枫斗类石斛的rDNA ITS序列数据库,寻找金钗石斛特异性的序列位点,设计专用于金钗石斛鉴别的PCR引物,用该引物在不同条件下扩增石斛属植物的模板DNA,建立只有金钗石斛具有阳性扩增的PCR鉴别条件。结果在66℃、40 s的复性条件下进行鉴别PCR,金钗石斛的模板DNA扩增出约300 bp的阳性扩增带,而其他39种石斛的模板DNA在同样条件下无扩增产物。结论运用位点特异性PCR能有效地从其他石斛属植物中鉴别出金钗石斛,该方法具有高效、准确、简便、省时的特点,应用前景广泛。

PCR技术检测食源性致病菌的研究进展

食源性致病菌的检测技术是食源性疾病预防与控制的关键环节。PCR是近年来广泛应用于食源性致病菌快速检测的方法之一。在食源性致病菌中,用于PCR检测的靶基因包括各种毒力基因、酶基因及特异性鉴别基因。这些靶基因的发现推动了食源性致病菌PCR快速检测的发展。

奶牛流产病因探究及新孢子虫病PCR检测方法的建立

【目的】探究新疆某奶牛场奶牛流产病因。【方法】采集的19份血清样品和4份牛流产胎儿脑组织进行布氏杆菌病凝集实验、弓形虫PCR检测和新孢子虫病ELISA方法检测,参考GenBank登录的犬新孢子虫种Nc-5序列[1],设计引物,建立新孢子虫病的PCR检测方法。【结果】调查结果显示该牛场布氏杆菌病与弓形虫病感染均呈阴性,新孢子虫病感染率为14.3%(2/19)。新孢子虫PCR诊断方法的最佳退火温度为57℃,灵敏度为35 pg/μL。用建立后的PCR方法检测流产胎儿,感染率为50.0%(2/4)。【结论】新孢子虫病是导致该厂奶牛流产的重要原因之一,为今后新疆地区有效预防和治疗新孢子虫病提供了一定的科学依据。

中药材鳖甲的位点特异性PCR鉴定研究

目的 本研究旨在建立一种简便、实用的鳖甲 DNA分子鉴定方法。方法 根据中华鳖和鳖甲混淆品原动物的 12 S r RNA基因片段的序列数据库 ,找出中华鳖与其它鳖科动物有明显区别的位点 ,设计 1对能特异性鉴别中华鳖的引物 ,然后利用鳖甲中残存的 DNA,通过 PCR扩增 ,即可准确鉴别鳖甲。结果 用这对引物对不同来源的10块鳖甲进行鉴定结果表明 ,其中有 3块为伪品 ,结果与 DNA序列分析鉴定一致。还测定了斑鼋和鳖甲一伪品12 S r RNA基因片段序列。结论 该引物配置药材鉴定试剂盒可在鳖甲类药材鉴定使用。